Indian Journal of Animal Research

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Full Research Article

Potential Impacts of Dietary Bacillus licheniformis Inclusion on Growth Performance, Serum Metabolites, Antioxidant Profiles and Immune Status of Broiler Chickens

M. Shawky1, G. Rayan2, Y. Alyousef2, H. Darrag3, H. Najib2, A. Mohammed2,*
1Avian Research Center, King Faisal University, P.O. Box 402, Al-Ahsa 31982, KSA.
2Department of Animal and Fish Production, College of Agriculture and Food Sciences, King Faisal University, P.O. Box 402, Al-Ahsa 31982, KSA.
3Research and Training Station, King Faisal University, P.O. Box 402, Al-Ahsa 31982, KSA.

ABSTRACT

Background: Probiotics have become increasingly important in poultry industry due to their potential beneficial impacts on improved gut health, enhanced growth performance and boosted immune system.

Methods: One hundred and sixty one day old Ross308 broiler chicks were allocated at random into four groups, with each group having four replicates of ten chicks. The groups were fed a basal diet containing Bacillus licheniformis (B. licheniformis) probiotic at concentrations of 0, 250, 500 and 1000 mg/kg feed. Feed intake (g), feed conversion (g/g) and body weight gain (g) were recorded. Dressing percentage and goblets parts were recorded upon slaughtering of control and B. licheniformis groups. Blood samples were centrifuged for serum collection and further analyses of antioxidant and metabolites profiles. Additionally, the antibody titer of the humral immune response against the Newcastle virus vaccine was evaluated using the ELISA test.

Result: The results of the current study showed that dietary probiotic B. licheniformis consumption improved (P<0.001) feed intake, feed conversion ratio and body weight gain. Dressing and goblets percentage remained unaffected by the dietary probiotic B. licheniformis incorporation if compared to control. Serum antioxidant and metabolites profiles were improved with the probiotic B. licheniformis incorporation. Additionally, significant differences between control, 250 B. licheniformis, 500 B. licheniformis groups compared to 1000 B. licheniformis group on antibody titer (p<0.05) against Newcastle. It could be concluded that adding 250 or 500 mg/kg of B. licheniformis probiotic to the feed significantly boosted broiler chick growth, improved their metabolic function and enhanced their antioxidant capabilities.

INTRODUCTION

The Earth’s population of the world is expected to reach 9.7 billion by the middle of the 21st century and 10.4 billion by 2100 (Gu et al., 2021; Mohammed and Alshaibani 2025; Mohammed et al., 2025). The increasing number of people on earth poses a serious threat to food security, requiring a substantial expansion of food production (Fróna et al., 2019). The need for increased food output requires us to adopt efficient and sustainable practices that minimize our environmental footprint (Wang et al., 2021). Consequently, we need to implement technological innovations in agriculture, minimize food waste and create fair distribution systems to guarantee sufficient food for a growing population (Mok et al., 2020; Khan et al., 2021).
       
Poultry is a highly efficient and inexpensive source of animal protein (Korver 2023; Alyousef et al., 2025). This makes it crucial for meeting the nutritional needs of a growing global population (Smith et al., 2024). It plays a vital role in combating malnutrition, especially in developing countries, by providing essential nutrients (Gržiniæ​ et al., 2023). Therefore, the incorporation of dietary probiotics in broiler chicken diets has gained significant attention due to its potential impact to enhance poultry production and health (Krysiak et al., 2021; Ayana and Kamutambuko, 2024). Studies have demonstrated that dietary probiotics significantly enhance broiler chicken performance, resulting in improved feed conversion, nutrient utilization, weight gain and gut health (Yousaf et al., 2022; Hu et al., 2024; Naeem and Bourassa, 2025).
               
Bacillus licheniformis (B. licheniformis) is gaining significant attention in poultry production due to its potential as a beneficial probiotic (Ramirez-Olea et al., 2022; Qin et al., 2024). The bacterium B. licheniformis contributes to a balanced gut environment by encouraging the proliferation of beneficial bacteria while suppressing harmful ones (Jin et al., 1997; Dumitru et al., 2024; Wongsamart et al., 2025). It can enhance the intestinal barrier function, reducing the risk of pathogen invasion (Kan et al., 2021; Han et al., 2023; Huang et al., 2023). B. licheniformis has been found to alleviate intestinal damage caused by conditions like necrotic enteritis (Xu et al., 2021; Xiao et al., 2024). By improving digestion and nutrient absorption, B. licheniformis can lead to increased body weight gain and improved feed conversion ratios (Qin et al., 2024). B. licheniformis produces various digestive enzymes, such as protease, amylase and lipase, which aid in the breakdown of feed (Shleeva et al., 2023). Consequently, B. licheniformis might offer a valuable tool for enhancing growth performance, careful consideration is essential for optimal results. Therefore, this study sought to determine the effects of the dietary probiotics (B. licheniformis) supplementation (0, 250, 500, or 1000 mg/kg feed) on growth performances, carcass traits, serum metabolites and biochemical profiles and Newcastle disease antibody titer of growing broilers.

MATERIALS AND METHODS

All experimental procedures were received ethical committee clearance of King Faisal University [KFU-REC-2025-MAR-EA252670]. The procedure of experiment was executed in the experimental animal research station of King Faisal University. B. licheniformis® is a probiotic feed additive containing of live spores of a distinct Bacillus licheniformis strain (Strain Identification Number DSM 28710) available commercially.
 
Site of study and experimental design
 
This study was conducted from November 2024 to January 2025. A total of one hundred sixty one-day old Ross308 broiler chicks were classified via random assignment over four groups (Fig 1). The basal diet’s composition adhered to the nutritional guidelines for the strain as recommended by Aviagen (2018). These birds were received the basal diet with added supplements at levels of 0, 250, 500, or 1000 mg/kg of Bacillus licheniformis, which were commercially available (B. licheniformis®) (control, 250 B. licheniformis, 500 B. licheniformis and 1000 B. licheniformis, respectively). The doses of B. licheniformis® were incorporated directly into the feed during mixing to guarantee homogeneity. The birds were raised in 16-square-foot floor pens. For the initial three days, the chicks were maintained at 33.0oC. Subsequently, the temperature was slowly lowered to 31.0oC over the following 48 hours. The birds were raised subsequently at the averaging 31.0oC natural ambient temperature and 60.0% relative humidity during the study. The lighting program was maintained at 23 h light to 1h dark for the first 7 days, followed by 18 h light to 6 dark for the remaining of the experiment. The chicks had ad libitum access to water and diets.

Fig 1: Experimental design of study for B. licheniformis ® additives to chicks.


 
Growth and carcass traits
 
Body weight gain (Kg) and feed intake (Kg) were measured at 7, 14, 21 and 28 days old on a pen basis and feed conversion ratio were calculated (Abdel-Moneim et al., 2025). On day 28 of age, eight chicks per group representing the groups’ average weight were chosen at random for carcass assessment to guarantee impartiality in the sample. The selected chicks were fasted for 12:0 h and weighed, then slaughtered, defeathered and their internal organs removed. The eviscerated carcasses and giblets were weighed of all groups. The eviscerated carcasses and giblets were recorded using digital balance to ensure accuracy and expressed as a percentage of live body weight (Abdel-Moneim et al., 2025).
 
Blood sample collection and analyses
 
Blood samples of control and B. licheniformis® groups were collected from the brachial vein (10 bird/group). The obtained blood samples were centrifuged to obtain serum for biochemical and antioxidant analyses (Analyzer Skyla VB1). The biochemical analysis encompassed total proteins, liver enzyme levels, urea, creatinine and mineral concentrations. The antioxidant enzymatic activities of serum malondialdehyde (MDA) was measured using a colorimetric method that involves its interaction with 2-thiobarbituric acid at a wavelength of 532 nm (Mihara and Uchiyama, 1978). The ferric reducing ability (FRAP) test was conducted as described by Benzie and Strain with modification (Benzie and Strain, 1996). The ABTS radical cation decolorization test was conducted as the recommendation of Re et al., (1999). The free radical scavenging activity (FRSA) was assessed as outlined by Blois with modification (Blois, 1958). Total thiol (sulfhydryl group, -SH) concentrations (TTL) were quantified as described by Hu with modification (Hu, 1994).
 
Blood samples and serological test of antibody titer against Newcastle disease
 
Blood samples (10 bird/group) were obtained by puncturing the Vena ulnaris and then transferred into 2 ml Micro-centrifuge Tubes. The clot blood samples were centrifuged for 3 minutes at 12,000 rpm for obtaining serum and stored at -20oC until use. The antibody level for Newcastle disease was detected using ELISA method on serum samples. The ELISA kits (ID Screen® Newcastle Disease REF NDVS-CV-5P LOT J36, France) were used to measure the antibody titer against Newcastle (Oberländer et al., 2020).
 
Statistical analysis
 
Body weight gain (g), feed conversion (kg/kg), dressing and giblets percentage (%), serum antioxidant and metabolites values of control and B. licheniformis® treated groups were statistically analyzed using SAS program (SAS 2008) through General Linear Model of one way ANOVA procedure according to the model:
 
Where,

μ = Mean.
Ti = Effects of B. licheniformis ® concentrations (250 B. licheniformis, 500 B. licheniformis  and 1000 B. licheniformis).
Eij = Standard error.
       
To compare the significant among means of control and B. licheniformis groups, Duncan’s multiple range test (1955) was used.

RESULTS AND DISCUSSION

The effects of B. licheniformis ® dietary inclusions (250, 500 and 1000 B. licheniformis® ) to Ross-308 broiler chicks on feed intake, body weight, feed conversion, carcass traits, serum metabolites and antioxidant capacity were shown in (Fig 2,3) and (Table 1,2).

Fig 2: Effects of B. licheniformis (250, 500 and 1000) on dressing and giblets percentage of broiler chicks at 28 d of age.



Fig 3: Effects of B. licheniformis (250, 500 and 1000) on newcastle disease antibody of broiler chicks at 28 d of age.



Table 1: Effects of B. licheniformis (250, 500, 1000 mg/kg) on feed intake, feed conversion and body weight gain of broiler chicks.



Table 2: Effects of B. licheniformis on body weight gain, feed intake and feed conversion of broiler chicks on plasma biochemistry profiles of broiler chickens at 28 d of age.


 
Growth performances
 
Changes of weekly body weight (g), weekly feed intake (g) and weekly feed conversion (kg/kg) are presented in (Table 1). Values of finally body weight (week 4) was significantly increased in 250, 500, 1000 B. licheniformis® groups compared to that of control group. Control group had the lowest values of body weight across all weeks compared to B. licheniformis® groups. B. licheniformis (500) group consistently had the highest values across all weeks compared to control and other B. licheniformis groups. Regarding to weekly feed intake, there is a clear trend of increasing feed intake across all treatments from week 1 to week 4. The control group showed the lowest feed intake in weeks 2, 3 and 4 compared to B. licheniformis groups. In addition, from week 2 to week 4, B. licheniformis (500) group consistently showed a significantly (P<0.003) higher feed intake compared to control and other B. licheniformis groups (250 and 1000). Concerning to feed conversion (kg/kg), a lower feed conversion ratio generally indicates better efficiency. In weeks 1, 2 and 3, there were no significant differences between any of the groups. In week 4, B. licheniformis groups (250 and 500) showed significantly better feed conversion ratios than control and B. licheniformis group (1000) was between them. Dressing and goblets percentage none significantly increased (P>0.05) by the dietary probiotic B. licheniformis incorporation if compared to control (Fig 2).
 
Serum metabolites and antioxidant capacity
 
The effects of B. licheniformis dietary inclusions (250, 500 and 1000 mg/kg diet) on serum metabolites and antioxidant capacity to broiler chickens were shown in (Table 1). The B. licheniformis dietary inclusions (250, 500 and 1000) resulted in higher total serum protein values compared to the control group. In 250 B. licheniformis group, albumin and creatinine showed the highest values (P<0.01) compared to the control and other B. licheniformis groups. Glucose values were not differed among groups (P=0.50). The B. licheniformis inclusions of all groups were decreased bilirubin values compared to control (P<0.0001). Regrading to AST and ALP, there were no significant differences in AST (P=0.74) and ALP (P=0.30) levels among the control and B. licheniformis groups. Regrading to ALT and GGT, there were significant decreased in ALT (P= 0.001) and GGT (P=0.03) in B. licheniformis 500 and B. licheniformis 1000 compared to control and B. licheniformis 250 groups. Regarding to antioxidant capacity, the FRAP, FRSA, ABTS and TTL values were not differed among control and B. licheniformis groups whereas MDA values were significantly (P = 0.01) decreased in B. licheniformis groups if compared to control group.

Newcastle disease antibody
 
Regarding to Newcastle antibody titer, the results showed significant differences between control, 250 B. licheniformis and 500 B. licheniformis groups compared to 1000 B. licheniformis group (p<0.05) on antibody titer against Newcastle (Fig 3).
       
The effects of B. licheniformis dietary inclusions (250, 500 and 1000 mg/kg diet) to Ross-308 broiler chicks on feed intake, body weight gain, feed conversion, carcass traits, serum metabolites and antioxidant capacity and Newcastle disease antibody are showed in (Fig 2,3) and (Tables 1-2). The results of the current study indicated the highest significant improvement of B. licheniformis@ dietary inclusions to Ross-308 broiler chicks on growth performance, serum metabolites and antioxidant capacity at level of 250 and 500 mg/kg. The effects of B. licheniformis could be attributed to several factors including improved feed utilization, gut health, boosted immune system and reduction of pathogens (Kan et al., 2021; Ramirez-Olea et al., 2022).
 
Growth performances
 
The effects of B. licheniformis dietary inclusions to broiler chicks on growth performances have been explored in several studies (Dumitru et al., 2024; Wongsamart et al., 2025).  Our results indicated that dietary 250 mg/kg B. licheniformis inclusion gave the highest body weight gain and feed conversion compared with 500 mg/kg and 1000 mg/kg B. licheniformis and control groups. B. licheniformis acts as a multi-faceted probiotic in broiler chicks, improving growth performance by enhancing digestion and nutrient absorption, promoting a healthy gut environment and balanced microbiota, stimulating the immune system and positively influencing metabolic functions and stress responses (Kan et al., 2021; Pan et al., 2022). These combined effects of B. licheniformis contribute to better feed conversion ratios, increased body weight gain and improved health and productivity in broiler chickens (Ramirez-Olea et al., 2022; Qin et al., 2024).
       
B. licheniformis produces various digestive enzymes like amylase, pectinase and cellulose, protease and lipase, which aid in breaking down complex feed components including carbohydrates, proteins and fats. This enhances nutrient digestibility and makes them more available for absorption by the chicks (Su et al., 2020, Shleeva et al., 2023; Sun et al., 2023). B. licheniformis consumes oxygen in the gut, creating an anaerobic environment that favors the growth of beneficial bacteria like Lactobacilli and Bifidobacteria (Ramirez-Olea et al., 2022; Dumitru et al., 2024). By consuming oxygen and competing for nutrients and colonization sites, B. licheniformis helps to inhibit the growth of harmful aerobic bacteria such as E. coli and C. perfringens (Abd El Hack et al., 2020). B. licheniformis supplementation can lead to a more balanced and diverse gut microbiota, which is crucial for overall health and nutrient absorption (Han et al., 2023).
 
Serum metabolites, antioxidant capacity and newcastle disease antibody
 
The values of serum metabolites and antioxidant capacity were improved in B. licheniformis groups compared to control group as in other studies (Xu et al., 2021; Yu et al., 2022). Total protein and albumin (g/dl) were significantly increased in B. licheniformis groups compared to control one. This could be attributed to the significant improvement of feed intake and feed conversion obtained in those groups, which subsequently resulting in increased of glucose and creatinine values. This could be attributed to the effect of B. licheniformis on gut microbiota. Total bilirubin values were decreased (P<0.0001) in B. licheniformis groups compared to control groups. B. licheniformis supplementation has shown potential to improve liver health in poultry.  By maintaining healthy liver function, these additives may contribute to efficient bilirubin processing and excretion in addition to lower liver enzymes (Table 1). This could be attributed to the effect of B. licheniformis on  modulation of gut microbiota and the gut-liver axis. The antioxidant capacity were decreased in B. licheniformis groups with increasing dose if compared to control group. B. licheniformis can produce various bioactive compounds with antioxidant properties as exopolysaccharides, certain amino acids, organic acids and phenols (Shleeva et al., 2023).
       
Immune response against Newcastle disease virus vaccine showing that 250 and 500 B. licheniformis groups similar to control group whereas1000 B. licheniformis group showed lower immune response. Additionally, all control and B. licheniformis groups were in protective level against Newcastle virulent virus. B. licheniformis can upregulate the gene expression of tight junction proteins and mucins, strengthening the intestinal barrier and reducing intestinal permeability.
       
This helps prevent the leakage of harmful substances and pathogens into the bloodstream (Obianwuna et al., 2023; Sun et al., 2023). Additionally, components of B. licheniformis such as cell wall polysaccharides and peptidoglycans, can act as antigens, stimulating the gut-associated lymphoid tissue and enhancing the immune response. Furthermore, studies have shown that B. licheniformis can increase the levels of immunoglobulins like IgA in the serum and intestinal mucosa, which are essential for defense against pathogens. It can activate macrophages and promote the production of T and B lymphocytes, enhancing the overall immune ability of the chicks (Yu et al., 2022; Qin et al., 2024).

CONCLUSION

It could be concluded that Bacillus licheniformis dietary inclusions modulates growth performances, serum metabolites and antioxidant capacity. The best dietary supplement levels were 250 B. licheniformis, followed by 500 and 1000 B. licheniformis levels, referring to the optimal amount of this additive to achieve maximum benefits and economic efficiency for poultry production. Determining the best dietary additive depends on poultry species and age, dietary composition and environmental conditions.

ACKNOWLEDGEMENT

The authors want to thank and acknowledge Deanship of Scientific Research, King Faisal University, Saudi Arabia for funding and support (KFU251590).
 
Disclaimers
 
The views expressed in this article are the authors’ alone and do not represent the opinions of their institutions.

Informed consent
 
Ethical approval for all experimental animal procedures and care methods was granted by the University’s Animal Care Committee.

CONFLICT OF INTEREST

No conflict of interest for authors to declare.

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