Research Article

Probiotic Powder Supplementation Enhances Ileal Villus Health and Systemic Immunity in Indigenous Chickens

Lovita Adriani1*, Ronnie Permana1, Mathew Piero Ngugi2, Andi Mushawwir1

1Animals Physiology and Biochemistry Laboratory, Department of Animal Nutrition and Feed Technology, Faculty of Anim. Husbandry, University of Padjadjaran, Jatinangor Campus Jl. Ir.Soekarno KM.21, Jatinangor-Sumedang 45363, Indonesia; 2Department of Biochemistry, Microbiology and Biotechnology, School of Pure and Applied Sciences, Kenyatta University, Kenya.

Received | November 10, 2025; Accepted | December 03, 2025; Published | December 22, 2025

*Correspondence | Lovita Adriani, Department of Animal Nutrition and Feed Technology, Animal Science Fac., University of Padjadjaran; Email: [email protected]

Citation | Adriani L, Permana R, Ngugi MP, Mushawwir A (2025). Probiotic powder supplementation enhances ileal villus health and systemic immunity in indigenous chickens. Adv. Anim. Vet. Sci., 13(12):2773-2780.

DOI | https://dx.doi.org/10.17582/journal.aavs/2025/13.12.2773.2780

ISSN (Online) | 2307-8316

Copyright: 2025 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

The local poultry industry is experiencing steady growth, particularly in Indonesia, where various poultry breeds are available. In the early 2000s, small and medium-scale local chicken farming involved about 1.35 million farmers, increasing to 2.15 million by 2024 (Aritonang et al., 2024). This growth has been driven by higher consumption from urban populations, partly fueled by the expanding restaurant sector. The main challenge in the local chicken industry is resistance to various pathogens, especially in traditional farming systems (Adriani et al., 2021, 2024) that lack intensive cages and modern feed practices. Using antibiotics to prevent and treat infections has become a significant issue, particularly as consumers increasingly demand organic, residue-free products. Many studies have shown that administering plant-derived flavonoid compounds (Aritonang et al., 2025) and essential oils from different extracts (Chen et al., 2023) can effectively combat pathogens. However, some researchers have reported low effectiveness of natural extracts (Muhammad et al., 2023; Mushawwir et al., 2023) in boosting immunity and fighting pathogens. Other research indicates that providing active compounds to poultry significantly impacts their health (Mushawwir et al., 2021a, b; Purwanti et al., 2024), although isolating these compounds requires specialised skills and can be costly (Rahmania et al., 2022; Nurfauziah et al., 2024).

Another practical, easy-to-implement alternative for traditional farmers is probiotics. Fermenting milk with lactic acid bacteria is a simple and affordable technology, long known and used by animal farmers (Firmansyah et al., 2024; Gurnin et al., 2024). Several studies have demonstrated the effectiveness of liquid probiotics on egg production (Gurnin et al., 2024; Adriani et al., 2021), with increases in daily output of up to 10% (Rosiyanti et al., 2025). Administering liquid probiotics also significantly improves haematological conditions related to immunity, including increases in leukocytes, lymphocytes, anti-inflammatory cytokine signals (IL-10), as well as inhibiting IL-6 (pro-inflammatory) (Adriani et al., 2024; Noda et al., 2018) and boosts in antibody levels in broilers (Adedeji et al., 2022). Research reported by Manin et al. (2024) indicates that administering liquid probiotics is effective in preventing Salmonella, with a 4% dose in the feed. However, liquid probiotics are reported to be difficult to administer to animals, and the microbial viability is prone to decline (Adriani et al., 2024).

The molecular mechanism behind the effects of probiotic administration has been demonstrated by Noda et al. (2018), who showed that lactic acid bacteria given to commercial laying hens respond well to increased signalling related to hormones that regulate inflammation. Increased interleukin-10 (IL-10) indicates that probiotics in laying hens can lower the rate of inflammation. Other studies show higher lymphocyte counts and increased activity of memory and dendritic cells in response to antibody stimulation (Mushawwir et al., 2021b; Alharthi et al., 2023). This suggests probiotic administration in commercial laying hens can reduce inflammation and cell death.

Probiotics appear to be very effective and efficient. Their application in local chickens needs to be investigated in more detail concerning their potential to stimulate immunity and prevent tissue damage, especially in the ileum. Adding probiotics to the powder is also a better technique. Preliminary test results indicate ease of application in livestock and good microbial resistance. Therefore, in the current study, the effects of probiotic powder on morphometric responses of the ileum and immunity in several local Indonesian chickens are demonstrated.

MATERIALS AND METHODS

Experimental animal and design

Four-week-old indigenous chickens with an average body weight of 455.75 g were used in this experiment. The chickens were divided into four treatment groups, each consisting of one hundred birds. The first group served as the control group without probiotic powder (PPw) added to their feed (IC0), while the second, third, and fourth groups received PPw at 2% (IC1), 3% (IC2), and 4% (IC3), respectively. Each treatment was replicated five times, with each experimental unit containing 20 chickens.

The experimental cages used were open system cages with litter flooring. The cage flooring was made of wood shavings to facilitate water absorption. Each experimental unit cage was 2 x 1.5 x 2.5 m. Microclimate conditions within the experimental cages were recorded throughout the experimental period. The average daytime temperatures and humidity in the experimental cages were 27°C and 75%, respectively, while at night they were 25.5°C and 73%, respectively.

Preparation of probiotics powder

This study used four bacterial consortia: Lactobacillus acidophilus, L. bulgaricus, Bifidobacterium bifidum, and Streptococcus thermophilus. They were cultured with 5% (v/v) inoculum in 250 mL of De Man, Rogosa, and Sharpe (MRS) medium for 24 hours at 37°C. The probiotic was prepared using fresh pasteurised cow’s milk, to which the four cultured bacterial consortia were added.

A total of 5% Maltodextrin DE 10-12 was added to the liquid probiotic. After thorough stirring, the mixture was placed in a vacuum oven at 40°C for 48 hours, yielding a probiotic powder. A comparison of pH, total bacteria and lactic acid bacteria before being made into PPw (liquid probiotic) and PPW, based on this study, is shown in Table 1.

Table 1: Level of pH and bacteria count in probiotic yoghurt.

Probiotics form	pH	Total bacteria (CFU/ml)	Total lactic acid bacteria (CFU/mg)
Liquid probiotics	4.21	6.63 x 107	4.82 x 107
Probiotics powder	4.24	4.45 x 107	4.34 x 107

PPw was added to the feed daily, before being given to the experimental chicken.

Basal ration

During this study, a basal diet was provided, consisting of the following feed ingredients: Coconut oil, fish meal, rice bran, yellow corn flour, soybean meal, coconut meal, and bone meal. The diet had the following composition: 1.47%, 19.61%, 4.90%, 50.02%, 15.69%, 7.55%, and 2.47%, respectively. The nutrient composition of the ration and the level of PPw addition are shown in Table 2.

 

Table 2: Nutrient composition of the basal diet and levels of probiotic powder (PPw) supplementation.

Nutrient and energy metabolism	Treatment
	IC0	IC1	IC2	1C3
Crude protein (%)	17.78	17.78	17.78	17.78
Extract ether (%)	7.42	7.42	7.42	7.42
Crude fiber (%)	5.41	5.41	5.41	5.41
Calcium (%)	0.22	0.22	0.22	0.22
Phosphorus (%)	0.22	0.22	0.22	0.22
Lysine (%)	0.88	0.88	0.88	0.88
Methionine (%)	0.22	0.22	0.22	0.22
Cysteine (%)	0.22	0.22	0.22	0.22
Valine (%)	0.15	0.15	0.15	0.15
Tryptophan (%)	0.20	0.20	0.20	0.20
Energy Metabolic (Kcal/kg)	2590	2590	2590	2590
Addition
PPw (%)	0	2	3	4

 

Feed and drinking water were provided ad libitum, administered every morning and evening. Feed was calculated based on weekly requirements to avoid excessive feed residue in the feeders without reducing feed consumption.

Sampling and analysis technique

Blood and tissue samples were collected from the experimental chickens at 19 weeks of age, before laying (20 weeks of age). During the PPw application period, before sampling for data analysis, health and physiological condition checks were routinely conducted to ensure that the treatment did not induce physiological stress. A 3 mL syringe was used to draw blood from the right wing vein, and a 3 mL tube containing EDTA was used to collect the blood sample. Blood plasma was separated by centrifugation at 3500 rpm for 5 min. The plasma was used to determine glucocorticoid hormone concentrations. At the same age, ileum tissue samples were also collected. The chickens were slaughtered, and physiological solution was immediately perfused through the ileum while a 3 cm segment was cut with sterile scissors. The ileum samples were immediately placed in sample bottles containing fixative solution until all tissues were completely submerged.

In this experiment, the Haematoxylin-Eosin (HE) method was used to assess ileal morphometry in experimental chickens. Sections of 5 microns thickness were cut with a microtome and fixed for 24 hours in 10% neutral buffered formalin (NBF). The samples underwent dehydration, cleaning, and blocking with a Leica TP 1020 tissue processor. Paraffin, mineral oil, and xylene were removed at room temperature for 15 min. The tissue sections were stained with HE according to standard protocols, and xylol was used to remove residual stain. The difference between apoptosis and necrosis is determined based on the morphology of stained cells. Apoptosis was characterised by cell shrinkage, chromatin condensation, and the formation of bubbles on the cell membrane (blebbing) without rupture. In contrast, necrosis was characterised by cell swelling and lysis. Observation under a binocular microscope allowed for morphometric analysis.

Data analysis

The analysis results were comprehensively organised in MS Excel 2023. The effect of treatment was analysed using analysis of variance (ANOVA) based on a completely randomised design and a precision level of 95% (α = 0.05). The difference in means between treatments was determined using Duncan’s multiple-range test at the 95% confidence level (α = 0.05).

RESULTS AND DISCUSSION

Effect of PPw on ileum morphometrics of indigenous chicken

This study presents the effects of adding PPw to the diet on ileum length, villi height, villi width, and the total number of villi in Table 3. Microscopic visualisation results are shown in Figure 1.

 

Table 3: Profile of the ileum of indigenous chicken supplemented with PPw (19 weeks old).

Treatment	Parameters
	Villi Height (µm)*	Villi width (µm)*	Total Villi*#	Ileum length (cm)
IC0	536.76±3.83a	82.63±1.19a	46.12±1.32a	73.22±2.42a
IC1	548.33±4.04b	89.19±1.62b	47.62±1.32a	77.18±2.53b
IC2	551.67±3.24c	92.63±1.14c	49.90±1.18b	84.45±2.25c
IC3	556.55±3.31d	94.32±1.05d	51.24±1.28c	88.05±2.17d

 

*Calculated using a microscope magnification of 4x, #number per microscope field of view (4x magnification). a,bDifferent superscript notation in the same column, indicating a significant difference (P<0.05).

 

Current research shows a consistent impact of PPw on the ileal villi profile, as illustrated in Table 3 and Figure 1. This effect is confirmed by a general increase in the number and size of ileal villi with higher treatment levels, evidenced by significant differences (P<0.05) between treatments from IC0 to IC3.

The IC3 treatment group had the longest ileum, measuring 88.05 cm (Table 3). Additionally, the ileum profile was more developed with IC3, as reflected in the height, width, and total villi, which were 556.55 µm, 94.32 µm, and 51.24, respectively. Higher levels of PPw also correlated with increased growth of the ileum and villi. These findings support the idea that including PPw in diets at levels up to 4% can effectively enhance digestive health and promote the growth of digestive tissues.

 

This research indicates that non-pathogenic microbes in PPw can improve the digestive system’s ecological environment from a physiological perspective. Several earlier studies show that active peptides produced by lactic acid bacteria in probiotics (Manin et al., 2024) enhance nutrient absorption (Firmansyah et al., 2024) and increase feed digestibility (Adedeji et al., 2022). Furthermore, increased feed intake and digestibility have been associated with growth in intestinal tissue and muscle mass (Mushawwir et al., 2021b; Ahmed-Farid et al., 2021).

Effect of PPw on the death of villi cells in indigenous chicken

Cell death naturally occurs alongside growth, metabolic activity, and the biological state of chickens. This study reveals varying patterns of ileal villi cell death depending on the levels of PPw administration. Table 4 presents counts of normal cells and cells undergoing cell death, including necrosis and apoptosis. Figure 2 presents HE-stained images illustrating ileal villi cell death.

The profile of ileal villi cell death shown in Table 4 demonstrates the effectiveness of PPw in reducing the number of ileal villi cell deaths, while maintaining or increasing the number of normal cells. Necrotic cells in the IC3 treatment group (Table 4 and Figure 2) were the lowest (51.26 in 1000 cells) (P<0.05) compared to other treatment groups. Similarly, the number of apoptotic cells showed that IC3 administration was the most effective in reducing apoptosis. Administration at the lowest level (IC1) significantly reduced necrosis and apoptosis (P<0.05) compared to the treatment group without PPw (IC0).

 

Table 4: Profile of ileal villi cells death of indigenous chicken supplemented with PPw (19 weeks old).

Treatment	Parameters*
	Number of necrosis cells**	Number of apoptotic cells**	Normal cell count**
IC0	153.25±2.64b	92.25±3.64a	781.78±5.77b
IC1	89.63±3.65b	54.53±4.26b	849.45±5.84c
IC2	63.73±4.36c	41.57±3.73c	878.73±6.26c
IC3	51.26±2.83d	32.73±2.83d	923.25±7.27d

 

Description: *Calculated with a field of view (100x magnification lens); **Number of cells in 1000 cells; different letter notations in the same column indicate a significant difference (P<0.05)

 

 

The addition of PPw at a concentration of 4% (IC3) was highly effective in reducing cell death and increasing the number of normal ileal villi cells. These results provide strong evidence of the competitive advantage of lactic acid bacteria over pathogenic bacteria that naturally inhabit the anterior colon. This prediction is firmly grounded in previous research showing that lactic acid bacteria can create an acidic environment (low pH) in the digestive tract, particularly in the small intestine, extending into the posterior part of the small intestine (Aritonang et al., 2025). Meanwhile, Gurnin et al. (2024) reported that an acidic intestinal environment inhibits the growth of pathogenic bacteria.

 

Table 5: Immunity marker levels of indigenous chicken supplemented with PPw in the diet (19 weeks old).

Treatment	Parameters
	Lymphocytes (102/mm3)	Neutrophils (102/mm3)	IL-6 (ng/dL)	IL-10 (ng/dL)	Glucocorticoid (ng/dL)
IC0	59.53±0.42a	8.64±0.46a	5.12±0.02a	2.64±0.14a	11.73±0.66a
IC1	60.54±0.64b	6.26±0.45b	3.01±0.11b	6.74±0.63b	7.26±0.25b
IC2	62.64±0.47c	5.78±0.15c	1.92±0.01b	8.63±0.21c	5.04±0.74c
IC3	62.75±0.36c	5.51±0.21c	1.01±0.03c	8.44±0.06c	4.83±0.19d

 

a,bDifferent superscript notations in the same column indicate differences (P<0.05). IL-6: Interleukin-6; IL-10: Interleukin-10

 

It is established that pathogenic bacteria can induce cell death by inhibiting the activity of ion receptors on the surface of villus cells (Mushawwir et al., 2024). Inhibition of ion channels plays a crucial role in cell death (Kharazi et al., 2022). Molecularly, the presence of pathogenic bacteria in the intestine triggers an increase in signals for cytokine hormone production (Muller et al., 2022; Mushawwir et al., 2023). Previous studies have shown a strong link between pro-inflammatory cytokine hormone levels and cell death, including both apoptosis and necrosis (Noda et al., 2018; Yaqoob et al., 2021).

Effect of PPw on immunity marker

The concentrations of various immunity markers resulting from PPW administration in this study are presented in Table 5. The table indicates that the immune response of the experimental chickens fed PPW varied, with a tendency to improve as PPW levels increased.

Lymphocyte levels increased with higher levels of PPw supplementation (Table 5). The experimental group with the highest IC3 supplementation had 62.75×10²/mm³ (P<0.05), compared with the other experimental groups with different PPw levels. The lowest supplementation level (IC1) was also higher (60.54×10²/mm³) than the group without PPw. These results suggest that PPw effectively boosts the immune response in chickens. It is known that lymphocyte levels can stimulate memory cells to produce antibodies. Tanuwiria et al. (2022a) reported that B lymphocytes help increase antibody levels by stimulating immunoglobulin production. Other studies have shown that high lymphocyte levels indicate strong immunity (Tanuwiria et al., 2022b; Petrilla et al., 2022).

Conversely, an increase in lymphocyte levels results in a decrease in neutrophil production (Table 5). These findings confirm that an enhanced immune response with PPw can counteract neutrophil activity. Neutrophils respond to the presence of pathogenic microbes or foreign proteins in the blood. The research results (Mushawwir et al., 2011, 2024; Chen et al., 2023) show a correlation between reduced pathogen infection and neutrophil levels. Other studies have also reported that an increase in lymphocytes is associated with a decrease in neutrophil levels, suggesting that immune potential is enhanced.

Immunity enhancement stimulation is characterised not only by the differentiation response of white blood cells but also by changes in specific protein levels within the cytokinin hormone group (Table 5). Administration of PPw significantly reduced IL-6, a pro-inflammatory signal. This is evident from the lower IL-6 levels after PPw administration in the IC3 experimental group (1.01 ng/dL) compared with the IC2, IC1, and especially the IC0 groups. Conversely, in the same treatment group, IL-10, an anti-inflammatory protein (8.44 ng/dL), increased significantly (P<0.05) compared to the other treatment groups. Previous studies have confirmed that increased inflammation is associated with elevated IL-6, and that, conversely, elevated IL-10 is associated with decreased inflammation (Nurfauziah et al., 2024; Chen et al., 2023).

Another interesting finding of this study was a reduction in glucocorticoid levels as PPw supplementation increased. The hormone level was 11.73 ng/dL in the IC0 experimental group and significantly decreased (P<0.05) to 4.83 ng/dL at the IC3 supplementation level. Overall, these results demonstrate a close link between cell death and glucocorticoid expression (Figure 3).

 

The presence of natural pathogens in the digestive tract not only directly causes cell death, but pathogenic bacteria here can also trigger pro-inflammatory cytokines, which are another factor contributing to high IL-6 levels without PPw administration (as shown in Table 5). At the same time, this signal also signals the adrenal medulla cells to secrete glucocorticoids (as shown in Table 5). The cytokine signal is also transmitted to tumour necrosis factor alpha (TNF-α) (Dudi et al., 2023; Petrilla et al., 2022). Increased TNF-α enhances glucocorticoid production (Tanuwiria et al., 2023; Mushawwir et al., 2021a; Zhao et al., 2023). This is the main reason for the positive correlation between total cell death and glucocorticoids (Figure 3), with a correlation coefficient of r = 0.85 and R² of 72.09%.

Previous research has shown that chemical signals from pathogenic proteins boost the activity of pro-inflammatory proteins (Muller et al., 2022). The expression of proteins involved in inflammation can lead to increased cell death (Alharthi et al., 2023). Additionally, an increase in glucocorticoids may serve as an indirect marker of cell death, as this hormone is closely linked to pro-inflammatory protein signalling (Noda et al., 2018). Metabolic changes and heightened inflammation, which help supply enough energy to maintain normal tissue homeostasis, also physiologically activate signalling pathways that promote glucocorticoid synthesis (Zhao et al., 2023; Gurnin et al., 2024).

CONCLUSIONS

The addition of PPw in this study was well tolerated by native chickens, as shown by the ileal villi profile and various immune indicators. The current results confirm that PPw at 4% (IC3) can significantly enhance ileal villus growth. Furthermore, PPw at this level appears highly effective in reducing cell death (apoptosis and necrosis) and increasing the number of healthy ileal villi cells. Simultaneously, the physiological effects induced by PPw at the IC3 level may boost immunity.

Based on these research results, it can serve as a reference for further research. Testing higher levels of PPw to determine its optimal addition. Additionally, a comprehensive study of its effectiveness using a multi-omics approach can be intensive, not only for indigenous chickens but also for other poultry species.

ACKNOWLEDGEMENTS

This research involved various parties. The author would like to express their gratitude to the staff of the Animal Physiology and Biochemistry Laboratory, Faculty of Animal Science, Padjadjaran University, for conducting the entire sample analysis. This research was also fully funded by a basic research grant from the Indonesian Ministry of Higher Education, Research, and Technology.

NOVELTY STATEMENT

Research on adding probiotics has been widely documented, but reports on using powdered probiotics are rare, especially in indigenous chickens. Specifically, studies on how probiotic administration affects ileal villi growth and immunity have not been reported. Traditionally raised indigenous chickens are vulnerable to pathogenic infections. Therefore, the strategy of adding PPw presents a highly innovative solution.

AUTHORS’ CONTRIBUTION

The design, implementation, and writing of the results of this research involved all authors listed in this article, each playing an equal role based on their expertise and capacity.

Ethical approval

The entire process of designing and conducting this research has been reviewed for ethical feasibility by the Animal Research Ethics Committee of the National Research Agency, with decision letter No. 963/KEP.07/SK/07/2025.

Generative AI and AI-assisted technology statement

All authors sincerely declare that the drafting and writing of this article did not use any applications related to artificial intelligence (AI).

Conflict of interest

All authors have declared no conflict of interest.

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