Bacillus-based probiotics on broiler chicken performance under coccidiosis and Clostridium perfringens challenge

 

 

Rafaela Berto^I; Nilton Rohloff Junior^I; Thiago dos Santos Andrade^{I, #}; Cristine Kaufmann^I; Heloisa Sartor^I; Ana Paula Guimarães Cruz Costa^I; Gabriel Natã Comin^I; Gabrielli Toniazzo^I; Vinicius de Queiroz Teixeira^II; Victor Daniel Naranjo^II; Cinthia Eyng^I; Ricardo Vianna Nunes^I

^IDepartment of Animal Science, Western Paraná State University, Marechal Cândido Rondon, PR, 85960-000, Brazil
^IIEvonik do Brasil, São Paulo, SP, 04711-904, Brazil

 

 

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ABSTRACT

The study analyzed the impacts of two probiotics (Ecobiol^® and GutCare^®) on growth performance, carcass and cut yields, intestinal morphometry and lesion score, biochemical parameters, and short-chain fatty acid concentration in chickens using a challenge model combining coccidiosis vaccine and Clostridium perfringens. A total of 880 one-day-old male broilers (Cobb 500) were randomly assigned to four treatments with 10 replicates and 22 birds per experimental unit (EU). The treatments consisted of: PC: positive control with the inclusion of 80 g ton^-1 of Enramax^® (8% enramycin) until 35 d of age; NC: negative control without the inclusion of growth-promoting probiotic; BA: NC with the inclusion of 1000 g ton^-1 of Ecobiol^® (Bacillus amyloliquefaciens - CECT 5940); BS: NC with the inclusion of 500 g ton^-1 of GutCare^® (Bacillus subtilis - DSM 32315). Diets used maize-soybean meal in a three-phase plan. Broiler chickens on the BA and BS diets had higher feed consumption, body weight gain, and improved feed conversion efficiency at 28 and 42 d when compared to the birds in the negative control. Broiler chickens fed PC, BA, and BS diets had a higher villus height and absorption area in the jejunum at 28 d, compared to the birds in the negative control. There was more butyric acid production by the intestinal microbiota at 28 d of age in broilers on the BA diet. Supplementing with 1000 g/ton of BA and 500 g/ton of BS effectively substituted the 8% enramycin antibiotic, enhancing broiler growth during an induced intestinal challenge.

Keywords: Antibiotic alternatives, intestinal health, poultry nutrition

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Introduction

Enteric diseases pose a considerable concern in the poultry industry, with necrotic enteritis being one of the most important, causing billions in annual losses (Fathima et al., 2022; Ningsih et al., 2023). The primary causative agent is Clostridium perfringens, which can produce toxins harmful to the gastrointestinal tract of birds (Hofacre et al., 2018; Ma et al., 2019; Ayalew et al., 2022; Rodrigues et al., 2024). The presence of this pathogen is exacerbated by factors such as changes in diet, imbalances in the intestinal microbiota, stress, and the occurrence of underlying diseases, such as coccidiosis caused by Eimeria spp. (Ahmed et al., 2014; Mwangi et al., 2019; Cirilo et al., 2023). High adaptability to various environments, tolerance to oxygen presence, the ability to survive in a wide pH range, and the ability to form spores make disease control challenging (Zhang et al., 2018; Liao et al., 2020; Ayalew et al., 2022; Lee et al., 2023; Zhang et al., 2023).

The use of antibiotics is a common alternative to control the spread of these enteric diseases in poultry farming (Gadde et al., 2017; Cai et al., 2023; Cirilo et al., 2023). However, indiscriminate antibiotic use can lead to the development of microbial resistance, making these treatments less effective over time (Bortoluzzi et al., 2019; Aljumaah et al., 2020; Madlala et al., 2021; Bao et al., 2022; Wang et al., 2023). Given this scenario, it has become essential to seek sustainable and effective alternatives for controlling enteric diseases in birds (Hernandez-Patlan et al., 2019; Gharib-Naseri et al., 2021; Memon et al., 2021; Zhu La et al., 2024). In this context, probiotics emerge as a promising alternative (Wang et al., 2021; Rahman et al., 2022; Chen et al., 2024). Specifically, Bacillus spp.-based probiotics have proven to be a viable solution, as they modulate the intestinal microbiota, optimize nutrient digestion, strengthen the immune system of broiler chickens, and reduce intestinal lesions caused by pathogens, such as Clostridium perfringens and Eimeria spp. (Cao et al., 2018; Adhikari et al., 2020; Ahmat et al., 2021; Goo et al., 2023).

Based on this, the hypothesis of this study is that probiotics derived from Bacillus may replace antibiotics in the diet of broiler chickens, resulting in improvements in performance and reduction of lesions caused by intestinal pathogens. Therefore, two probiotics (Ecobiol^® with Bacillus amyloliquefaciens - CECT 5940 and GutCare^® with Bacillus subtilis - DSM 32315) were used as alternatives to enramycin to evaluate the growth performance, carcass yield and cuts, intestinal morphometry, lesion score, biochemical parameters, and short-chain fatty acid concentration in broiler chickens challenged with Clostridium perfringens and Eimeria vaccine.

 

Materials and methods

The use of animals and approval for all experimental protocols were granted by the Ethics Committee for Animal Use of Western Paraná State University (Protocol number: 20/2020). The study was conducted at the Poultry Research Center of Western Paraná State University, Marechal Cândido Rondon, Paraná, Brazil.

A total of 880 one-day-old male broiler chickens (Cobb 500) with a mean weight of 43.48 ± 0.55 g were distributed in a completely randomized design with four treatments and 10 replicates of 22 broilers per pen. The treatments were composed of: PC: positive control with the inclusion of 80 g ton^-1 of Enramax^® (8% enramycin) until 35 d of age; NC: Negative control without the inclusion of growth-promoting probiotic; BA: NC with the inclusion of 1000 g ton^-1 of Ecobiol® (Bacillus amyloliquefaciens -CECT 5940); BS: NC with the inclusion of 500 g ton^-1 of GutCare® (Bacillus subtilis - DSM 32315). The dosage of the probiotics was adjusted to meet the specific criteria of the experiment, ensuring their effectiveness in the study conditions.

The Enramax^® used in this study is a formulation containing 8% enramycin, recognized for its specific efficacy in the digestive tract microbiota against Clostridium perfringens, Staphylococcus, and Streptococcus, contributing to the preservation of intestinal integrity. The Ecobiol^®, also used in the research, consists of a natural strain of Bacillus amyloliquefaciens - CECT 5940, acknowledged for its ability to stabilize the intestinal flora and promote intestinal microbial balance. GutCare® used in the study is also recognized for maintaining this intestinal microbial balance with a natural strain of Bacillus subtilis - DSM 32315.

The diets were formulated based on maize and soybean meals according to the nutritional requirements established by Rostagno et al. (2017), for the respective age phases: 1-21 d (starter), 22-35 d (grower), and 36-42 d (finisher). Broilers received feed, in mash form, and water ad libitum throughout the experimental period. The use of the test products (Enramax® - enramicina 8%, Ecobiol^® and GutCare^®) was done on weight-for-weight (g g^-1) replacement for the inert feed material (kaolin).

 

Table 1

 

At day 4 of age, all broilers were challenged with 20 times the dose of the Biococcivet R^® vaccine (concentrated suspension of sporulated oocysts of five species of Eimeria: E. acervulina, E. praecox, E. maxima, E. tenella, and E. mitis), i.e., they received 0.6 mL orally to damage the intestine to facilitate colonization of Clostridium perfringens. At 7 and 10 d of age, all broilers received a 0.5 mL solution of culture inoculum with Clostridium perfringens (10E8 CFU mL^-1) directly into the oesophagus in the region near the crop. This Clostridium perfringens strain was obtained from a field sample isolated from an outbreak of necrotic enteritis in broilers.

The poultry were housed in a 25-m-long by 8-m-wide concrete-floor barn, divided into pens of 1.76 m² each, equipped with nipple-type drinkers and tubular feeders and 250-watt heating lamps. The environment featured negative pressure ventilation and evaporative cooling for climate control. Before the experiment, the concrete floor of these pens was covered with pine shavings. The lighting program followed the recommendations of the breeder manual (COBB 500, 2019). Temperatures and relative humidity were monitored daily.

During the experimental period, mortality was recorded daily to correct feed intake and feed conversion, following the methodology described by Sakomura & Rostagno (2016). At 28 and 42 d of age, both feeders and birds were weighed to determine feed consumption, body weight gain, and feed conversion rate of the birds.

At 42 d of age, two broilers per experimental unit were randomly selected, weighed, marked, and slaughtered. The carcasses were weighed and stripped, obtaining legs (thigh and drumstick), breast fillet and inner breast fillet, which were weighed individually. Carcass yield was determined by the eviscerated carcass weight in relation to the live weight of the poultry. The cut yield was determined by the ratio between the weight of the cuts and the weight of the eviscerated carcass. The liver and abdominal fat (consisting of the adipose tissue around the cloaca, gizzard, proventriculus and adjacent abdominal muscles) were separated and weighed to determine their weight relative to the live weight of broilers.

At 28 d of age, two broilers per experimental unit were randomly selected for blood sample collection via brachial puncture on the ulnar vein while in lateral recumbency. After the collection, the birds were returned to their respective experimental units. Specific vacuum blood collection tubes made of 13 × 75 mm glass with clot activator and 5 ml capacity (CRAL, São Paulo, Brazil), specific adapters and 25 × 0.8 mm needles (Labor Import, Shandong Weigao, China) were used to collect 4 ml of blood per bird. After collection, the samples remained horizontal for 15 min and then were centrifuged (Kasvi brand, K14-4000, Paraná, Brazil) at 2500 rpm (1,050 × g) for 10 min at room temperature (20-25 °C). After serum separation, they were identified and packed in 2 ml microtubes (CRAL, São Paulo, Brazil) and stored in a freezer at -20 °C until the analysis was performed (Nunes et al., 2018). To perform the analyses, the samples were thawed under refrigeration (4 °C), remaining in the refrigerator for 24 h. Before performing the analyses, the samples were centrifuged in a microcentrifuge (Eppendorf® brand, Minispin®, Hamburg, Germany) at 1,050× g for 10 min at room temperature to remove the possible presence of fibrin. The measurements of the biochemical parameters were performed using an automatic biochemical analyser with spectrophotometry using the Flexor EL200 (Elitech^® brand, Flexor EL200 model, Puteaux, France), using reagents, calibrators (Elical II) and calibration standards (Elitrol I) from Elitech®.

The parameters evaluated were: uric acid, performed based on the Trinder enzymatic colorimetric endpoint method (Trinder, 1969); glucose, using the Trinder enzymatic colorimetric kinetic method (Trinder, 1969); cholesterol using the Trinder enzymatic colorimetric endpoint method (Allain et al., 1974); triglycerides using the enzymatic colorimetric endpoint method (Fossati and Prencipe, 1982); total protein, using the biuret endpoint method (Rifai et al., 2018); albumin using bromocresol green (BCG) colorimetric method (Doumas and Biggs, 1972; Wu, 2006); and creatinine using the Jaffe colorimetric kinetic method (Rifai et al., 2018). The determinations of the enzymatic activities for aspartate aminotransferase (AST) using the IFCC method without pyridoxal phosphate were performed using a kinetic assay and were measured with an ultraviolet spectrophotometer to monitor the absorption changes during the reaction (Schuman et al., 2002a); alanine aminotransferase (ALT) activity was determined using the IFCC method without pyridoxal phosphate, using a kinetic assay and measured with an ultraviolet spectrophotometer (Schuman et al., 2002b); gamma glutamyltransferase (GGT) using the glupa C substrate method, kinetic (Schuman et al., 2002c); lactate dehydrogenase (LDH) using the IFCC method, kinetic (Schumann et al., 2002d); creatine phosphokinase (CPK) using the IFCC method, kinetic (Schumann et al., 2002e).

At 28 d of age, one bird per experimental unit was randomly selected and euthanized for intestinal morphometry analysis. Crypt depth, villus height, absorptive area, and the ratio of villus height to crypt depth were evaluated. The small intestine was exposed, and both the duodenum and the jejunum were separated for sampling. In the duodenum, the segment considered ranged from the pylorus to the distal portion of the intestinal loop, whereas in the jejunum, it extended from the distal portion of the duodenal loop to the Meckel's diverticulum. A 2-cm fragment was collected from the duodenum in the ascending portion of the duodenal loop, and a 2-cm fragment was collected from the jejunum, 5 cm before the Meckel's diverticulum.

This fragment was fixed in 10% buffered formalin solution, dehydrated in increasing ethanol concentrations, and embedded in paraffin. Subsequently, semi-serial sections with a thickness of 5 μπι were made from each segment, mounted on glass slides, and stained using the haematoxylin-eosin technique as described by Luna (1968). Following slide preparation, the length and width of 10 villi, as well as the depth and width of 10 crypts, were analysed using the PROPLUS IMAGE 5.0 imaging system. These morphometric measurements were used to calculate the absorptive surface area of the intestinal mucosa, using the formula proposed by Kisielinski et al. (2002). Additionally, the ratio of villus height to crypt depth was calculated by dividing the value of villus height by the value of crypt depth.

At 28 d of age, macroscopic lesions of Clostridium perfringens and Eimeria spp. were evaluated in broilers that were sacrificed to perform the intestinal morphometry analysis. Lesions caused by Eimeria spp. and Clostridium perfringens were assessed following the lesion scoring system established by Johnson & Reid (1970): Grade 0 - no lesions, Grade 1 - mild lesions, Grade 2 - moderate lesions, Grade 3 - severe lesions, and Grade 4 - very severe lesions.

One broiler per experimental unit was slaughtered at 28 d of age for collection of the cecal contents according to the methodology of Souza et al. (2022). The cecal contents were removed and 200 mg were weighed and transferred to 2-ml individually marked microtubes (CRAL, Cotia, Sao Paulo, Brazil). A solution of 1800 μ! of 1% (w/v) NaOH was added, which was vigorously homogenized in a multifunctional vortex (Kasvi Brand, K40-1010, Sao José dos Pinhais, Paraná, Brazil) for 2 min at 3,000 rpm.

After homogenization, the microtubes were centrifuged (Kasvi Brand, K14-4000, Paraná, Brazil) at 1,050 × g for 5 min for complete sedimentation of the solid fraction of the sample. A total volume of 900 μl of the supernatant was transferred (Single-channel Micropipette Plus 100-1000 Kasvi, K1 -P1000, Paraná, Brazil) to new microtubes (CRAL, Cotia, Sao Paulo, Brazil) and was acidified with 50 μl (Single-channel Micropipette Plus 10-100 Kasvi, K1 - P100, Paraná, Brazil) of 50% (w/v) ortho-phosphoric acid solution. The acidified samples were homogenized in a vortex (Kasvi Brand, K40-1010, Sâo José dos Pinhais, Paraná, Brazil) for 30 s at 3,000 rpm and stored in a freezer at -20 °C until the readings were taken.

The concentrations of acetic, propanoic, butyric, valeric, and isovaleric acids in the samples were determined using gas chromatography using a Shimadzu® GC-2010 Plus chromatograph equipped with AOC-20i automatic injector, Stabilwax-DA™ capillary column (30m, 0.25mm DI, 0.25μηΊdf, Restek®), and flame ionization detector (FID), after acidification with 1 M o-phosphoric acid p.a. (Ref. 100573, Merck®) and fortification with a mixture of free volatile acids (Ref. 46975, Supelco®). An aliquot of 1 μL of each sample was injected with a split ratio of 40:1, using helium as carrier gas with a linear velocity of 42 cm s^-1, obtaining the separation of the analytes in a chromatographic run of 11.5 min.

The injector and detector temperatures were 250 °C and 300 °C, respectively, and the initial column temperature was 40 °C. The temperature ramp of the column started with a gradient from 40 to 120 °C at a rate of 40 °C min^-1, followed by a gradient from 120 to 180 °C at a rate of 10 °C min^-1, and from 180 to 240 °C at a rate of 120 °C min^-1, keeping the temperature at 240 °C for another 3 min at the end. For the quantification of the analytes, a calibration method was performed with dilutions of the standard WSFA^-2 (Ref. 47056, Supelco®) and glacial acetic acid (Ref. 33209, Sigma-Aldrich®) analysed under the conditions described above. Peak determination and integration were performed using GCsolution v. 2.42.00 software (Shimadzu®). The results were expressed in mmol kg^-1.

Upon completion of the experiment, normality was assessed, followed by an analysis of variance. In cases of significant effects, the Tukey test was used with a 5% probability for comparing means. For data that did not meet the normal distribution criteria, a non-parametric Kruskal-Wallis analysis was conducted, and if a significant effect was observed (P <0.05), treatments were compared using Dunn's test at 5%. These analyses were conducted using SAS software (SAS, 2014).

 

Results and Discussion

Broilers fed the treatments BA (Bacillus amyloliquefaciens - CECT 5940), BS (Bacillus subtilis -DSM 32315) and PC (enramycin antibiotic) improved (P = 0.001) weight gain and feed conversion ratio in 28-d (Table 2) and 42-d (Table 3) broilers, compared to broilers fed the NC diet.

The lower feed conversion ratio indicated that a reduced amount of feed was required to reach 1 kg of live weight (de Oliveira et al., 2019). These results highlight a greater efficiency of broiler chickens in utilizing nutrients for muscle growth, contributing to an improvement in carcass yield in the groups of birds that received probiotics, which showed similar results to the PC group (enramycin). The increase in weight gain of broiler chickens fed with the probiotics BA (Bacillus amyloliquefaciens - CECT 5940) and BS (Bacillus subtilis - DSM 32315) can be attributed to the ability of these bacterial strains to promote better digestion and absorption of nutrients in the gastrointestinal tract of the birds (Ma et al., 2018; Ningsih et al., 2024). Additionally, these strains may have contributed to a greater balance of the intestinal microbiota, favouring beneficial bacteria, and inhibiting the growth of pathogens (Menconi et al., 2020; Sun et al., 2022), which reduces competition for nutrients, decreases gastrointestinal disorders, and increases the efficiency of feed conversion into body mass. These factors result in faster and healthier weight gain of broiler chickens during the growth period (Wang et al., 2023; Rodrigues et al., 2024).

Other studies have also observed favourable outcomes in performance when evaluating the use of probiotics based on Bacillus subtilis - DSM 32315 and Bacillus amyloliquefaciens - CECT 5940 in the diets of broilers challenged with necrotic enteritis. These studies suggest improved nutrient absorption, enhanced energy utilization by the birds, and greater antioxidant capacity, humoral immunity, and increased activity of endogenous gastrointestinal enzymes (Hernandez-Patlan et al., 2019; Whelan et al., 2019; de Oliveira et al., 2019; Menconi et al., 2020; Gharib-Naseri et al., 2021; Sun et al., 2022; Larsberg et al., 2023).

Broilers fed with the PC, BA, and BS treatments exhibited higher carcass yields (P = 0.001), fillet yield (P = 0.001), yield of sasami (P = 0.001), and relative weight of abdominal fat (P = 0.001) in comparison to chickens fed with the NC diet. However, the birds fed with NC demonstrated higher breast fillet yield (P = 0.001), wing yield (P = 0.016), and relative liver weight (P = 0.001) when compared to the other treatments (Table 4).

The relative liver weight was lower in the poultry supplemented with probiotics, suggesting that there was a reduction in inflammation in the organ caused by necrotic enteritis, improving the resistance of birds to the disease. Corroborating with this study, Ramlucken et al. (2020) when evaluating the effect of a multi-strain bacillus (CPB 011, CPB 029, HP 1.6, and D014) probiotic on broilers challenged with Clostridium perfringens, observed that treatment without the probiotic promoted an increase in relative liver weight, suggesting that subclinical Clostridium perfringens infection caused inflammation of the liver.

According to Immerseel et al. (2004), it is possible that a high number of Clostridium perfringens bacteria in the small intestine of poultry pass into the bile ducts and through the portal circulation to reach the liver. Therefore, the current study suggests that supplementation with B. subtilis DSM 32315 and Bacillus amyloliquefaciens CECT 5940 in poultry diet may have a positive impact on reducing liver inflammation caused by necrotic enteritis, particularly following the challenge. This is likely achieved through the regulation of the intestinal microbiome and prevention of pathogen dissemination to other organs (Ma et al., 2018; Larsberg et al., 2023). This approach contributes to promoting overall health and disease resistance in the birds (Zhang et al., 2023; Zhu La et al., 2024).

The different responses of breast fillet, wing yield, and relative weight of abdominal fat may be associated with the metabolic changes caused by using the probiotics and the antibiotic, providing changes in the poultry caecal contents and amino acid metabolism, and reducing protein deposition in the muscle (Cao et al. 2018). The higher fat position in broilers supplemented with probiotics may be related to increased feed intake and weight gain (Abdel-Raheem and Abd-Allah et al., 2011; Wang et al., 2021; Ningsih et al., 2023).

The probiotic (BA and BS) did not affect any of the blood parameters (P >0.05), such as albumin, cholesterol, creatinine, glucose, total protein, triglycerides, and uric acid (Table 5). Probiotic (BA and BS), PC, and NC did not affect (P >0.05) alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, gamma glutamyltransferase, and creatinine phosphokinase content (Table 6).

In the analysis of duodenal intestinal morphometry in 28-day-old chickens (Table 7), the PC, NC, and diets containing probiotics (BA and BS) showed no effect (P >0.05) on crypt depth and the ratio between villus height and crypt depth. Villus height was altered in birds that received diets with probiotics (BA and BS) and with the antibiotic (PC) (P = 0.001). Additionally, it was observed that crypt width was greater in birds fed BA (P = 0.001), whereas villus width was greater in birds that received the probiotic, BS (P = 0.001).

In the analysis of jejunal intestinal morphometry in 28-day-old chickens (Table 8), the probiotic (BA and BS) and PC diets influenced villus height (P = 0.001), villus height to crypt depth ratio (P = 0.040), and absorption area (P = 0.001). Probiotic (BA) provided greater change in villus width (P = 0.003), crypt depth (P = 0.001), and crypt width (P = 0.023) when compared to probiotic BS and PC diets.

The intestinal morphometry of the duodenum and jejunum in 28-day-old birds suggests that probiotics derived from Bacillus hold promise as a substitute for antibiotics in broiler diets (Ningsih et al., 2023; Rodrigues et al., 2024). This substitution not only enhances performance but also reduces lesions caused by intestinal pathogens (Wang et al., 2023; Zhang et al., 2023). The observed changes in intestinal morphometry parameters suggest that probiotics can trigger structural modifications in the intestine, potentially boosting nutrient absorption, immune function, and overall intestinal health (Memon et al., 2021 ; Osho et al., 2023).

These findings deepen our comprehension of the mechanisms behind the beneficial impacts of probiotics in poultry farming and advocate for their adoption as antibiotic alternatives to bolster intestinal health and performance in broilers (Poudel et al., 2022; Rodrigues et al., 2024). This metabolic improvement likely occurred through various mechanisms, including competition with pathogenic bacteria for nutrients and space, production of antimicrobial substances such as organic acids and bacteriocins, and modulation of the immune response in the intestine, ultimately resulting in improved intestinal health and performance (Xu et al., 2021; Zhu La et al., 2024).

Other studies corroborate these findings, reinforcing the potential of probiotics as an alternative to antibiotics in poultry production (Mazanko et al., 2022; Cirilo et al., 2023; Wang et al., 2023). They consistently demonstrate improvements in intestinal morphology, nutrient utilization, and immune responses in birds (Cirilo et al., 2023; Zhang et al., 2023). Additionally, they highlight a substantial reduction in the occurrence of intestinal diseases (Lee et al., 2023; Osho et al., 2023; Chen et al., 2024).

Lesion score in broilers fed the NC diet and infected at 28 d with Eimeria spp. increased (P = 0.001) to 1.30 (presence of streaks or petechiae on the mucosa) (Table 9). This condition was recovered with supplementation of the probiotics (BA and BS) and the antibiotic enramycin, substantially improving the gut lesion score (score 0). For the treatments with feed additives, no differences were detected between enramycin and the probiotics.

The reduction in intestinal lesions was possibly related to the decrease in coccidiosis oocysts in the presence of probiotics (BA and BS), reducing damage to the epithelium and the leakage of nutrients into the lumen. Typically, enteric infections are caused by pathogens that dominate the mucosal surface in the animal's intestine, disrupting the balance of the microbiota (Abd El-Hack et al., 2022; Larsberg et al., 2023).

The use of probiotics (BA and BS) enabled the exclusion of pathogens through competition and reduced the severity of intestinal lesions on the mucosa, as reflected in the intestinal morphometry of the duodenum and jejunum, where greater villus height and absorption area were observed compared to the negative control, maintaining the integrity and functionality of the epithelial barrier (Liao et al., 2020). These findings emphasise the efficacy of probiotics in promoting a healthier and infection-resistant intestinal environment, resulting in substantial benefits for the health and performance of poultry (Cirilo et al., 2023; Rodrigues et al., 2024).

Other studies support the findings of the present study, emphasising the importance of probiotics, particularly Bacillus strains, in playing a crucial role in reducing intestinal damage and enhancing the overall health and performance of poultry (Wang et al., 2023; Zhang et al., 2023; Ningsih et al., 2024). In the study conducted by Farhat-Khemakhem et al. (2018), it was observed that the Bacillus amyloliquefaciens US573 strain exhibited high adherence to the intestinal mucosa and the capability to form biofilms. These characteristics provide protection against pathogens, possibly forming a barrier that prevents the infective form of Eimeria spp. from invading enterocytes and causing damage to the intestinal mucosa. Additionally, Wang et al. (2021) investigated the benefits of Bacillus subtilis in minimizing the negative effects of necrotic enteritis in broiler chickens, noting a reduction in intestinal lesions and an increase in the ratio between villus height and crypt depth. The probiotic also induced a marked decrease in the expression of the Muc-2 gene, potentially reducing mucus secretion and, consequently, diminishing the availability of nutrients for Clostridium perfringens and its adhesion to the mucosa, providing a preventive effect against necrotic enteritis.

Broilers supplemented with the diet containing the probiotic, BA (Table 10) exhibited a higher concentration of butyric acid in the cecum (P = 0.004). There was no difference observed between the treatments for the other tested short-chain fatty acids (P >0.05).

The BA probiotic modulates the intestinal microbiota, favouring the proliferation of beneficial bacteria and increasing butyric acid production, known for its positive effects on intestinal health, including the suppression of inflammatory diseases and improvement of immune function (Ayalew et al. 2022; Kouhounde et al., 2022). Previous studies highlight the crucial role of probiotics containing Bacillus strains in regulating the intestinal microbiota, promoting an environment conducive to the growth and development of birds (Zhang et al., 2018; Liao et al., 2020; Xu et al., 2021; Mátis et al., 2022).

The presence of Bacillus amyloliquefaciens in the BA probiotic may favour the colonization of Lactobacilli and Bifidobacteria, which in turn contribute to butyric acid production (Qaisrani et al., 2015). These findings are consistent with the observation of a reduction in intestinal lesions and an improvement in intestinal morphology in birds supplemented with the BA probiotic (Ma et al., 2019; Hong et al., 2019; Whelan et al., 2019; Cirilo et al., 2023). Furthermore, the increased presence of butyric acid may confer resistance to intestinal pathogens, such as Clostridium perfringens and Eimeria spp., contributing to the prevention of gastrointestinal diseases in broiler chickens (Timbermont et al., 2011; Li et al., 2018; Bao et al., 2020; Sun et al., 2022; Larsberg et al., 2023).

Therefore, the results of this study indicate that probiotics BA (Bacillus amyloliquefaciens - CECT 5940) and BS (Bacillus subtilis - DSM 32315) outperform the NC diet (without probiotics or antibiotics) and yield comparable results to enramycin (PC). This superiority of probiotics over the NC diet suggests that these bacterial strains play a crucial role in optimizing bird performance (Cai et al., 2023; Wang et al., 2023; Rodrigues et al., 2024). Probiotics enhance nutrient digestion and absorption, as demonstrated by the studies of Ma et al. (2018) and Ningsih et al. (2024), while balancing the intestinal microbiota by promoting beneficial bacteria and inhibiting pathogens, as evidenced by Menconi et al. (2020) and Sun et al. (2022). The similarity between probiotic results and the PC diet (enramycin) suggests that probiotics can effectively replace antibiotics as growth promoters, reducing the risk of antimicrobial resistance and ensuring bird performance and intestinal health (Osho et al., 2023; Chen et al., 2024; Zhu La et al., 2024).

 

Conclusion

The use of Ecobiol® (Bacillus amyloliquefaciens - CECT 5940) at a rate of 1000 g ton¹ and GutCare® (Bacillus subtilis - DSM 32315) at a rate of 500 g ton¹ in the diets of broiler chickens has proven to be an effective strategy to replace the antibiotic, Enramax® (8% enramycin). This administration resulted in performance and carcass yields similar to those obtained with the use of Enramax®. Moreover, these Bacillus-based probiotics promoted improvements in the intestinal health of broiler chickens, without affecting serum metabolites.

 

Acknowledgements

Not applicable.

Author contributions

Rafaela Berto, Nilton Rohloff Junior, Cristine Kaufmann, Heloisa Sartor, Ana Paula Guimarães Cruz Costa, Gabriel Natã Comin: investigation, methodology, data curation, formal analysis, software, and project administration; Vinicius de Queiroz Teixeira, Victor Daniel Naranjo, Cinthia Eyng, Ricardo Vianna Nunes: conceptualization, methodology, and project administration, supervision, validation, and visualization; Thiago dos Santos Andrade: writing - review and editing. All authors have read and agreed to the published version of the manuscript.

Conflict of interest

The authors declare no competing interests.

Ethics approval

The protocol of this research was in accordance with the Brazilian Normative Act No. 37, from February 15, 2018, by the national animal experimentation control board.

 

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Submitted 21 February 2024
Accepted 3 September 2024
Published 31 October 2024

 

 

# Corresponding author: thiagoandradefoz@hotmail.com