Effects of feeding eubiotics as antibiotic substitutes on growth performance, intestinal histomorphology and microbiology of broilers

Abstract

The inclusion of prebiotics and/or probiotics in broiler diets as a substitute for antibiotics is a topic of interest in poultry nutrition research due to concerns about antibiotic resistance and the need for sustainable poultry production practices. A total of 200 one-day-old chicks were housed in 40 cages, with eight cages repeated per treatment. Birds were fed a basal diet supplemented with one of five feed treatments at the following rates: 0 (control), 0.05 g Neoxyval/kg (antimicrobial growth promoter (AGP)), 0.2 g GalliPro^®/kg (probiotic), 0.6 g TechnoMos^®/kg (prebiotic) and a mixture of 0.2 g GalliPro^®/kg + 0.6 g TechnoMos^®/kg (symbiotic). During 0–42 days, the growth performance of broilers, carcass characteristics and intestinal histomorphology were investigated. The AGP, prebiotic and probiotic-treated birds outperformed the control and symbiotic-treated groups in terms of body weight gain, feed conversion, performance index and feed efficiency. The birds given AGP and probiotics had the longest partial and total small intestine length, while the birds given prebiotics had the smallest. In addition, prebiotic-treated birds had longer ileum villi and higher ileal lactic acid bacteria colonies than control and AGP-treated birds. Clostridium perfringens was eradicated from the ileum by probiotics, but not from the caecum. In conclusion, probiotics and prebiotics can be used as an alternative to antibiotics in broiler diets.

Highlights

• In terms of performance indices, the prebiotic and probiotic groups outperformed the control and symbiotic groups and performed comparably to the AGP group.

• Improvement of ileal histomorphology associated with increase of ileal absorption area and suppression of ileal pathogens, promoting feed efficiency of broilers.

• Prebiotics played a role in eradicating Clostridium perfringens as one of the pathogenic bacteria of the intestine.

Keywords:

• Antibiotic replacements
• broiler performance index
• probiotic
• prebiotic
• symbiotic

Introduction

The use of antibiotics as AGP in broiler chickens has been a common practice at subtherapeutic doses in the poultry industry for several decades to promote growth performance by improving feed efficiency (FE) and reducing the incidence of diseases (Paul et al. 2022). However, overuse and misuse of antibiotics in livestock farming have led to the development of antibiotic-resistant bacteria, posing a significant threat to human and animal health (Cella et al. 2023). Consequently, many countries have implemented regulations to restrict the use of antibiotics in animal agriculture (Emeje et al. 2022; Da Silva et al. 2023; Schmerold et al. 2023). As a result, there has been a growing interest in finding viable replacements for antibiotics as growth promoters in broiler chickens (Ahammed and Rahman 2022; Tukaram et al. 2022; El-Ghany and Wafaa 2024). Prebiotics, probiotics, symbiotics, phytogenics, enzymes, organic acids and other feed additives are now used in the poultry industry (Ahammed and Rahman 2022; Kalia et al. 2022; Yadav et al. 2022). A first possible alternative to AGPs are probiotics. Probiotics are beneficial live microorganisms that can improve the gut health of broiler chickens because of their antimicrobial and growth promoter abilities. By promoting a balanced gut microbiota, probiotics enhance digestion and nutrient absorption, leading to better growth rates (Ahammed and Rahman 2022).

Probiotics can also inhibit the growth colonisation of harmful pathogens in the intestines such as Salmonella spp and Clostridium perfringens by competitive exclusion mechanism (Kulkarni et al. 2022).

A second possible alternative to AGPs is prebiotics. Prebiotics are non-digestible oligosaccharides substances, produced by the animal’s enzymes that selectively promote the growth and replication activity of beneficial bacteria species in the gut, potentially improving the host’s health. By providing a favourable environment for beneficial microorganisms, prebiotics enhance gut health, improve nutrient utilisation and support the immune system.

Prebiotics provide greater advantages than probiotics because, unlike probiotics, they are intended to selectively encourage the beneficial microorganisms that already exist in the intestines (Yang et al. 2009; You et al. 2022).

Symbiotics are a third viable option for AGPs. Symbiotic is a probiotic and prebiotic combination that synergistically enhances the survival and colonisation of beneficial bacteria in the gut. Symbiotics offer a dual approach to improving diet digestibility and gut health and can be effective in promoting growth in broiler chickens (Kulkarni et al. 2022; Abed et al. 2023).

Probiotics, prebiotics and symbiotics, we hypothesise, can successfully substitute antibiotics as growth boosters and contribute to overall sustainability and safety in broiler chickens. This research aimed to compare prebiotics (TechnoMos^®), probiotics (GalliPro^®) and their combination (symbiotics) effects on broiler chicken productive performance during 42 days of age as well as on jejunum and ileum histomorphology to a standard AGP (Neoxyval).

Materials and methods

Birds management and experimental diet

The growth experiment was conducted from 1 to 42 days of chick age with the poultry experimental unit, Animal Production Department, King Saud University (KSU), Saudi Arabia, in line with Saudi Arabian legislation and a protocol approved by KSU. A total of 200 one-day-old Ross 308 males’ chicks were obtained from a local hatchery and distributed among 40 cages (8 replicates/treatment, 5 chicks in each replicate) in a four-deck cage system outfitted with electrically warmed battery brooders with elevated wire floors. Each cage has three dimensions: 50 × 60 × 36 cm.

Starter (0–14 d) and finisher (15–42 d) experimental diets were prepared and formulated in mash form based on maize-soybean meal. The testing products were placed on top (Supplementary Table S1).

The inclusion rates of five nutritional regimens for broilers were listed below:

1. Basal diet as control (non-supplemented basal diet);

2. Basal diet + 0.05 g antibiotics/kg (Neoxyval ‘AGP’);

3. Basal diet + 0.2 g probiotics/kg (GalliPro^®);

4. Basal diet + 0.6 g prebiotics/kg (TechnoMos^®);

5. Basal diet + symbiotic (0.6 g prebiotic/kg + 0.2 g probiotic/kg).

Avian infectious bronchitis, Newcastle disease and Marek’s disease vaccines were all given to the chicks. For the first three days, vitamins were added to the drinking water. The birds were raised in cages under similar management and sanitary circumstances, were fed and watered ad libitum, and were kept on 24-h light program. The ambient temperature was set at 33 °C on day 0 and gradually lowered to 22 °C by the rest of the trail. Relative humidity and ambient temperature were continuously and concurrently recorded at three-hour intervals using data loggers inside the chamber. The average relative humidity and temperature for the whole period were 26.6 ± 3.30% and 25.0 ± 0.26 °C, respectively.

The reference antimicrobial growth promoter (AGP) was Neoxyval, which contains 200 mg/g neomycin and 200 mg/g oxytetracycline. The antibiotic, probiotic, prebiotic and symbiotic products were obtained commercially by Biochem Zusatzstoffe Handels- und Produktionsgesellschaft mbH (Küstermeyerstraße, Germany). GalliPro^® is a probiotic that contains a minimum concentration of 1.6 × 10⁹ live spores/g of Bacillus subtilis (DSM 17299). TechnoMos^®, a prebiotic product, has prebiotic activity generated from the baker’s yeast Saccharomyces cerevisiae cell wall, which is rich in mannanoligosaccharides (MOS) and β-1,3-glucans.

Measurements

In pens, body weight and feed intake were recorded weekly, and feed conversion ratio (FCR), performance index (PI = bodyweight gain/feed conversion ratio) and FE were calculated. Mortality was tracked throughout the study and recorded daily if it occurred, and dead bird weights were used to modify FCR.

At the end of the experiment (42 days), eight broilers were selected per group. After euthanasia, the liver, breast, legs and abdominal fat were taken for each carcass and weighed. The percentage yield of each part and dressed yield were calculated based on live weight.

In addition, the small intestine of each slaughtered bird was aseptically removed. Jejunum, ileum and jejunum + ileum weights and lengths were measured. Intestine relative weight (IRW; g/100 g BW) was calculated. For histological measurements, 2 cm long specimens were taken from the proximal part of each small intestinal region. Specimens were fixed in phosphate-buffered formalin for at least 48 h before embedding in paraffin. The 5 µm sections were cut and stained with haematoxylin and eosin. An Olympus IX71 inverted microscope and a PC-based image digital camera (Olympus NV, Aartselaar, Belgium) with cellSens digital imaging software analysis were used.

Digesta contents of the ileum and caecum were emptied, and 1 g of each sample was weighed into a new glass on ice and diluted in 0.9% saline under aseptic conditions. To count the examined bacteria, 0.1 mL of each specimen was plated out in duplicate with selective media (Al-Sagan and Abudabos 2018). Clostridium perfringens was counted on tryptose sulfite-cycloserine (TSC) agar (Oxoid CM587) (Al-Sagan and Abudabos 2018; Kevenk et al. 2022). After incubating for 24 h at 37 °C in an aerobic condition, Enterobacteriaceae (Gram-negative bacilli; GNB) were isolated on MacConkey agar (Oxoid CM7) and identified by inoculation API 20E strips (bioMérieux Vitek, Marcy l’ Etoile, France) at 37 °C for 18 h after the wells were filled with the bacterial suspensions (Al-Sagan and Abudabos 2018). The results were recorded and expressed as log₁₀ colony forming units/g (log₁₀ CFU/g) of each sample (Al-Sagan and Abudabos 2018). To investigate carbohydrate utilisation by microorganisms, carbohydrate fermentation was tested using the API 50 CHL system. Strains of lactobacillus and related genera were identified based on carbohydrate fermentation and determined using the API 50 CHL kit in conjunction with API 50 CHL medium based on company manufacturing instructions (Biomerieux, Marcy l’ Etoile, France) (Truc et al. 2023). Marks (+/–) according to the colour change resulting from the reaction of the API strips. Custom software was used to identify species classifications.

Statistical analysis

ANOVA was used to analyse the data for a completely randomised design (CRD) applying SAS software’s general linear model approach (SAS, 2003, Cary, NC). Fisher’s protected test was used when the ANOVA revealed significant differences. p < .05 was chosen as the general statistical significance level. All values were expressed as means with a standard error of the mean (SEM).

Results

Performance indicators

Performance indicators are presented in Table 1 and Figures 1 and 2. During the cumulative period (0–42 d), treatments had an impact on performance indicators (p < .01). However, no statistically significant (p > .05) variations in feed intake were found. The result is likely to be explained by the tiny amount of feed consumed throughout the trial. The AGP, prebiotic and probiotic groups gained more weight than the control group (58.8, 56.7, 55.7 and 50.1 g/d, respectively) (p < .01), while the symbiotic group (52.2 g/d) was intermediate and not substantially different from the probiotic and control groups. The birds in the AGP, probiotic and prebiotic supplemented diet converted feed more efficiently (1.62, 1.65 and 1.65 g/g, correspondingly), while birds in control obtained the worst FCR (1.76 g/g) and then symbiotic (1.72 g/g) (p < .05). The order of treated groups based on FCR results was: control > symbiotic > probiotic ≥ prebiotic ≥ AGP.

Figure 1. Performance index of broilers fed experimental diets during d 0 to d 42. ^abcMeans with different superscripts differ significantly (p < .05).

Figure 2. Feed efficiency (gain/feed intake) of broilers fed experimental diets during d 0 to d 42. ^abMeans with different superscripts differ significantly (p < .05).

Table 1. Bodyweight gain (BWG), feed intake (FI) and feed conversion ratio (FCR) of broilers fed experimental diets during d 0 to d 42.

	Measurements
Treatment^1	BWG, g/d	FI, g/d	FCR, g/g	Mortality rate, %
Control	50.1^c	88.5	1.76^a	1.43
AGP	58.8^a	95.4	1.62^b	0.95
Probiotic	55.7^ab	91.9	1.65^b	0.95
Prebiotic	56.7^a	93.5	1.65^b	1.55
Symbiotic	52.2^bc	89.7	1.72^ab	0.48
SEM±^2	1.42	2.12	0.017	0.34
p Value	**	ns	**	ns

Means in the same column with different superscripts differ significantly (**p < .05).

¹ Birds were fed a basic meal supplemented with one of five dietary treatments at inclusion rates of 0 (control), 0.05 g Neoxyval/kg (antimicrobial growth promoter (AGP)), 0.2 g GalliPro^®/kg (probiotic), 0.6 g TechnoMos^®/kg (prebiotic) and 0.2 g GalliPro^®/kg + 0.6 g TechnoMos^®/kg (symbiotic).

² SEM: standard errors of means.

The AGP, prebiotic and probiotic groups had a higher PI than the control group (36.3, 34.4, 33.8 and 28.5, respectively) (p < .01), while the symbiotic group (30.3) was intermediate and not substantially different from the probiotic and control groups (Figure 1).

Feed efficiency was higher in the AGP, probiotic and prebiotic-supplemented meals (0.62, 0.61 and 0.61 g/g, respectively) than in the symbiotic (0.58 g/g) and control (0.57 g/g) diets (p < .05) (Figure 2). The results demonstrated that broilers fed a non-supplemented diet consistently performed poorly.

Carcass characteristics

The mean proportion of carcass components as a percentage of live weight for each treatment is revealed in Table 2. Treatment had no influence on relative weights (% BW) of breast, legs, abdomen fat or dressing yield % (p > .05). Diet, on the other hand, had an effect on liver weight relative to live weight. When compared to the control, birds given AGP and symbiotics had the highest liver weight.

Table 2. Effects of different treatments on dressed yield and relative weight (g/100 g of live body weight) of carcass parts of broiler at day 42.

Treatment^1	Liver	Breast	Legs	Abdominal fat	Dressed yield
Control	2.14^d	25.7	31.5	2.38	73.01
AGP	2.63^a	28.9	31.5	2.14	74.85
Probiotic	2.36^c	28.5	31	2.12	73.48
Prebiotic	2.54^ab	28.2	31.4	1.92	73.66
Symbiotic	2.58^a	28.1	31.1	2.03	74.12
SEM±^2	0.12	1.31	1.01	0.21	0.59
p Value	*	ns	ns	ns	ns

^abcdMeans in the same column with different superscripts differ significantly (*p < .05, ns: not significant).

¹ Birds were fed a basic meal supplemented with one of five dietary treatments at inclusion rates of 0 (control), 0.05 g Neoxyval/kg (antimicrobial growth promoter (AGP)), 0.2 g GalliPro^®/kg (probiotic), 0.6 g TechnoMos^®/kg (prebiotic) and 0.2 g GalliPro^®/kg + 0.6 g TechnoMos^®/kg (symbiotic).

² SEM: standard errors of means.

Small intestine morphology measurements

The small-intestine measurements are shown in Table 3. Dietary treatments had an effect (p < .01) on jejunal length, ileal length and jejunal plus ileal length. Birds administered AGP and probiotic had the longest jejunal, ileal and jejunal plus ileum lengths, whereas birds given prebiotic had the shortest; nevertheless, other groups had equal lengths. On the other hand, the weight and thickness of the jejunal, ileal and small intestine (jejunal plus ileal) and IRW were not influenced (p > .05) by treatment.

Table 3. Jejunum and ileum measurements at 42 d of age of broiler chickens fed different dietary treatments.

Treatment^1	Control	AGP	Probiotic	Prebiotic	Symbiotic	SEM^2±	p Value
Jejunum weight, W, g	18.9	21.4	19.1	18.7	18.7	0.585	.582
Jejunum length, L, cm	69.8^ab	77.3^a	76.2^a	65.5^b	70.5^ab	1.23	.005
Jejunum thickness, W/L, g/cm	0.271	0.276	0.251	0.285	0.265	0.006	.574
Jejunum weight, % LBW	0.825	0.902	0.895	0.923	0.874	0.022	.704
Ileum length, L, cm	64.5^b	74.3^ab	75.2^a	65.5^ab	72.3^ab	1.30	.008
Ileum weight, W, g	15.3	19.8	18.0	15.5	15.2	0.637	.068
Ileum thickness, W/L, g/cm	4.44	3.97	4.25	4.28	4.77	0.152	.575
Ileum weight, %LBW	145	148	169	191	166	11.6	.757
Jejunum + ileum length, cm	134^b	152^a	151^a	131^b	143^ab	2.25	.002
Jejunum + ileum weight, g	34.2	41.2	37.2	34.2	33.9	1.14	.200
Jejunum + ileum thickness, W/L, g/cm	0.253	0.270	0.245	0.260	0.237	0.006	.407
IRW, g/100 g BW	1.49	1.75	1.74	1.69	1.58	0.044	.297

IRW: intestine relative weight.

^a,bMeans in the same row with different superscripts differ significantly (p < .05).

¹ Birds were fed a basic meal supplemented with one of five dietary treatments at inclusion rates of 0 (control), 0.05 g Neoxyval/kg (antimicrobial growth promoter (AGP)), 0.2 g GalliPro^®/kg (probiotic), 0.6 g TechnoMos^®/kg (prebiotic) and 0.2 g GalliPro^®/kg + 0.6 g TechnoMos^®/kg (symbiotic).

² SEM: standard errors of means.

Small intestine histology measurements

Figure 3 depicts the height and diameter of the jejunal and ileal villus intestines. Treatment had an effect on ileal villi height (p < .05). Ileal villi were longer (p < .05) in the prebiotic (493 µm) group compared to the probiotic (440 µm) and control (425 µm) groups. There were no significant variations in ileal villi length between probiotic (455 µm), symbiotic (445 µm) and prebiotic (493 µm). Treatment, on the other hand, had no effect on jejunal villi length (p > .05). Dietary interventions had no effect on jejunal or ileal villus width (p > .05).

Figure 3. Intestinal histology of broilers fed experimental diets at 42 d old (p < .01).

Intestinal bacterial colonisation

Figure 4 shows data on bacterial counts in the ileal and caecum of broilers. The number of GNB in the ileum and caecum was similar in all groups (p > .05), indicating that the drug used in this experiment had no effect on GNB. The probiotic-treated birds successfully eliminated Clostridium perfringens from the ileum, but they did not statistically differ from the symbiotic-treated birds. Conversely, dietary therapy had no effect on the number of Clostridium perfringens in the caecum (p < .05). The number of lactic acid bacteria in the ileum increased significantly in the prebiotic groups (p < .05), but not in the drug-treated groups, while the control group had the lowest number. Lactic acid bacteria increased significantly in the treated groups compared to the control group (p < .05).

Figure 4. Gram-negative bacilli and Clostridium perfringens count, as well as lactic acid generation in broiler ileum and caeca at 42 days old (mean, log₁₀ CFU/g). ^abcMeans with different superscripts differ significantly (p < .05).

Discussion

The use of AGP in poultry feeding results in resistant microbial populations and consequently compromises consumer health. Alternative therapies for common poultry diseases have been extensively researched. Alternatives to antibiotics include eubiotics, which include probiotics, prebiotics and their combinations. Here, the addition of probiotics and prebiotics increased the body weight gain (BWG), FCR, PI and FE of broiler chickens, similar to AGP. Several studies have found that broiler performance with eubiotics is comparable to AGP (Al-Sagan and Abudabos 2018; Hussein et al. 2020; García-Reyna et al. 2023), although these substitutes do not insurance the economic and production benefits of an antibiotic (Murshed and Abudabos 2015; García-Reyna et al. 2023). The outcomes of this study with the tested probiotic are consistent with prior publications. Hussein et al. (2020) found that supplementing broilers with probiotics and phytobiotics, alone or in combination, can improve performance, gut health and blood components of broilers under Clostridium perfringens infection.

Bacillus probiotics are widely used in the chicken industry because they can promote growth performance and production efficiency, protect against pathogenic microorganisms, strengthen bones and help control parasite infections (Bahaddad et al. 2023). It becomes dangerous for both people and poultry when broilers are exposed to certain pathogenic microorganisms, increasing the risk of contamination at various points along the food chain. Therefore, Bacillus probiotics can act as a natural alternative against pathogenic germs such as Clostridium perfringens, Escherichia coli, Salmonella spp and Campylobacter jejuni at the pre-harvest stage (Jeong and Kim 2014; Ahasan et al. 2015; Mingmongkolchai and Panbangred 2018; Shaffi and Hameed 2023). Too, to avoiding colonisation of the gut by the pathogen, feeding poultry the probiotic Bacillus can increase FCR and improve gain. Moreover, the probiotic Bacillus exhibited positive effects on the intestinal histomorphology of broiler chickens, such as increases in villus height and villus height to crypt height ratio (Bahaddad et al. 2023). These benefits help to improve digestibility and increase bowel absorption capacity for nutrients (Jayaraman et al. 2013). Broilers given Bacillus enzyme-producing probiotics had lower digestive viscosity as a result of their capacity to benefit insoluble non-starch polysaccharides (NSPs), which are known to reduce nutritional availability and absorption (Sen et al. 2012). Several mechanisms have been proposed to explain why probiotics improve growth performance. Probiotics have been shown to inhibit the colonisation of the gut by harmful bacteria through the process of competitive exclusion (La Ragione and Woodward 2003; Abudabos et al. 2013). The ability of desirable probiotic bacteria to perform competitive exclusion/antagonism activities with undesirable intestinal pathogens for attachment to intestinal surfaces (Vieco-Saiz et al. 2019).

The prebiotic utilised in this study boosted bird’s performance to levels comparable to AGP. Asif et al. (2022) observed that birds fed diets containing prebiotic MOS had better production performance compared to control groups. Thus, prebiotics may replace AGPs as non-microbial performance enhancing feed proponents. While probiotics aim to introduce helpful microorganisms into the intestine, prebiotics may work by selectively activating the beneficial bacteria that currently exist. Prebiotics provide energy, metabolic substrates and vital micronutrients to the host by serving as fuel for the endogenous microbiota (Yang et al. 2009; Bhadra and Banerjee 2020; Grozina et al. 2023; Joseph et al. 2023). The addition of prebiotics improved broiler growth and FCR (Asif et al. 2022; Rahmani Alizadeh et al. 2023; Rousseaux et al. 2023). Several processes are thought to explain why prebiotics improve performance and health of broilers These mechanisms comprise decreasing the incidence of disease by preventing pathogenic bacteria from colonising the intestinal mucosa, proliferating and producing toxins, strengthening the immune system, reducing the number of intestinal pathogens, and improving the morphofunctional properties of the gut (Benites et al. 2008; Ajuwon 2016; Markowiak and Śliżewska 2018; Leone and Ferrante 2023).

Symbiotics are a mixture of prebiotics and probiotics (Rousseaux et al. 2023). Usually, prebiotics provide substrate for fermentation, improving probiotic organism viability. Symbiotic supplements (prebiotics plus probiotics) are often more effective than the separate supplements (Li et al. 2008; Mookiah et al. 2014). Likewise, Awad et al. (2009) indicated that a symbiotic outperformed a probiotic in avian performance, but this was not the case in the present investigation. When used together, prebiotics and probiotics can have a synergistic effect. While it is generally rare to observe antagonistic effects between prebiotics and probiotics, it is essential to consider the specific formulations and concentrations used. In some cases, excessive prebiotic supplementation might lead to overgrowth of certain pathogenic bacteria and depress beneficial bacteria population numbers, which shifts the gut microbiota to a state of dysbiosis.

In agreement with Abd El Latif and Omar (2023), the percentage of liver weight increased significantly in broilers in symbiosis. In contrast to Silva et al. (2018), the use of antibiotics (avilamycin and colistin), probiotics (Bacillus subtilis), prebiotics (mananoligosaccharides) and organic acids (fumaric acid and propionic acid) had no effect on liver, heart or intestinal weight. In addition, Hu et al. (2022) found that symbiotic supplementation as an alternative to antibiotics had no effect on liver weight. The result of the percentage improvement in liver yield with the addition of AGP in broiler diets could be related to the improvement in nutrient utilisation and better utilisation of feed digestion and absorption.

Intestinal tract size and structure are useful indicators for predicting the effects of dietary components on organ function and development in broilers (Moon et al. 2022). The longer the intestine, the more energy is required to maintain it, and ultimately, the lower the energy expenditure for production activities. In addition, the longer residence time of the digestive fluid required for the digestive process in the intestine further increases the length of the intestinal tract (Marume et al. 2020). Nutrient digestibility and FE are increased with reduced gut weight and length (Reis et al. 2017). However, in this study, it was found that the probiotic and AGP groups had greater gut length than the other groups, and FE increased with greater gut length. However, Moon et al. (2022) found that the probiotic group (Paenibacillus konkukensis) showed reduced gut length compared to the AGP group (enramycin), with no differences in growth performance or FE.

At day 42, histological analysis showed a substantial difference in villus height between treatments. Birds receiving eubiotics had enhanced ileal villus height, which was better than that of the control and AGP-treated birds. These findings suggest that villi morphology changed between groups at 42 d, increasing the absorption area in the intestine. Longer villi in poultry are usually associated with high absorption effectiveness and a healthier digestive tract (Pelicano et al. 2005). Maximum absorption and digestive capacity are ensured by a large lumenal area with villus height and mature enterocytes, which are essential for broiler development (Pelicano et al. 2005). Greater villus height and better development of ileal mucosa in broilers fed a yeast cell wall of Saccharomyces cerevisiae (Zhang et al. 2005). In addition, Salim et al. (2013) stated that the height and width of the ileal villus improved when broilers received probiotics. Asif et al. (2022) reported that supplementation with MOS showed positive effects on villi height and crypt depth in both jejunum, and ilium. Abdel-Raheem et al. (2012) observed that broiler chickens fed with symbiotics had higher villi height in the duodenum, jejunum and ileum than those fed with prebiotics and probiotics. In agreement with Oliveira et al. (2008), who observed that antibiotic supplementation reduced ileal villi height, this was attributed to the suppressive influence of the antibiotic on intestinal beneficial bacteria like Bifidobacteria and Lactobacillus. Brummer et al. (2010) indicated that there were no differences between Saccharomyces cerevisiae cell wall product treatments when villi width and height were measured. Wilson et al. (2005) postulated that pathogenic bacteria restrict growth by producing toxic compounds that irritate the intestinal mucosa, thereby reducing food absorption. The probiotic-treated group performed equally to the antibiotic-treated group in the current study, indicating that the probiotic studied can replace antibiotics in the diet without affecting broiler performance. The number of GNB was the same in the ileum and caecum in all groups, suggesting that the antibiotics and eubiotics tested in this study had no effect on GNB. As indicated by Bedford (2000), greatest AGPs target gram-positive microbes, suggesting that this group of microbes is leading to performance disorders. In this study, the anticlostridial properties of the selected products were tested. Clostridium perfringens is one of the major causes of necrotic enteritis, leading to health problems for birds if antibiotics are discontinued without replacing them with medications that restrict the multiplication of this bacterium, resulting in decreased digestion and absorption and subsequent poor performance (Bansal 2020). In addition, the addition of Bacillus subtilis PB6 to broiler chickens, which can produce an antimicrobial factor, reduced the numbers of many bacteria, including Clostridium perfringens, through the immunomodulatory effect of Bacillus subtilis (Yurong et al. 2005; Olson et al. 2021). The growth-inhibitory effect of intestinal pathogenic bacteria has been clarified by a toxin produced by pathogenic bacteria that can irritate the intestinal mucosa and restrict food absorption. As noted by Feighner and Dashkevicz (1987), the high degree of bile salt hydrolase action in Clostridium perfringens causes growth depression. The results indicate that administration of probiotics, either alone or as part of a symbiotic relationship, inhibited the propagation of Clostridium perfringens in the ileum. Clostridium perfringens (0.1 log₁₀ CFU/g) was eradicated by the probiotic in the ileum but not in the caecum. This eradication is attributed to competitive exclusion and immune stimulation.

When the prebiotic was given with meals, the ileal Clostridium perfringens population of the birds did not change. This contradicts the findings of Yang et al. (2007) and Spring et al. (2000), who found that prebiotics have a beneficial effect on reducing Clostridium perfringens populations. The ability of prebiotics to reduce Clostridium perfringens has been explained by their ability to control pathogens by providing competitive binding sites for unwanted pathogens (Kulkarni et al. 2022). The improvement in BWG, FCR, PI and FE, on the other hand, could be due to a reduced bacterial population in the digestive tract of birds (Thongsong et al. 2008).

In the small intestine, prebiotics are indigestible. As a result, bacteria attached to prebiotics are likely to leave the intestine without connecting to the intestinal epithelia, resulting in a reduction or prevention of unwanted bacterial colonisation (Spring et al. 2000).

Lactic acid bacteria, considered beneficial bacteria in the gut, increase in the eubiotics groups compared to the control and AGP groups. This observation is consistent with previous findings. According to Teo and Tan (2006), Bacillus subtilis produces acetic and lactic acids from glucose. The large increase in lactic acid colonies in the caecum and ileum in the groups supplemented with prebiotics and probiotics is further evidence that prebiotics and probiotics can alter the intestinal flora. Probiotics include yeast cells and bacterial cultures, both of which motivates microbes capable of restoring a healthy intestinal environment and enhancing FE. Probiotics alter the intestinal flora by promoting the growth of non-pathogenic bacteria that produce lactic acid and suppressing the propagation of intestinal pathogens.

Conclusions

In conclusion, broilers fed the probiotic (GalliPro^®) and prebiotic (TechnoMos^®) individually performed equally to those given the antibiotic (Neoxyval); however, the symbiosis did worse than either product alone and similarly to those given the control. Thus, probiotics (GalliPro^®) and prebiotics (TechnoMos^®) can be used to replace AGP in broiler feed to improve performance and health. As a result, we recommend using GalliPro^® or TechnoMos^®, but not both, in poultry meals. The increase in broiler performance could be ascribed to an increase in ileal height and ileal villus length, which is associated with intestinal pathogen suppression, resulting in an increase in ileal absorptive area and enhanced gut health.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data presented in this study are available on request from the corresponding author upon reasonable request.

Additional information

Funding

This work was supported by the Researchers Supporting Project (RSPD2023R1081), at King Saud University, Riyadh, Saudi Arabia.