Introduction

Gastrointestinal diseases are a major concern in the poultry industry because they negatively impact birds’ performance, mortality rates, and overall health1. The poultry intestine hosts a dynamic ecosystem that includes various microorganisms, which are influenced by several factors such as genetics, nutrition, and the environment2. Recent studies have focused on modifying the intestinal microbiota of birds to promote beneficial bacteria, such as lactobacilli. Research has confirmed that using additives like probiotics, prebiotics, and synbiotics can effectively modulate the intestinal microbiota of poultry3,4,5. One solution to this problem is to simultaneously use probiotics and prebiotics (synbiotics) in the diet. This hypothesis was strengthened since it was observed in some experiments when a probiotic is also used in diets containing prebiotics, the effectiveness of probiotics increases6. It has been reported that synbiotics in the broilers’ diet can produce a similar improvement as the growth-promoting antibiotics7. Enterococcus faecium (E. faecium) is a Gram-positive, non-spore-forming, facultative anaerobic bacterium. These bacteria can grow over a wide range of temperatures and pH. Some studies show the effect of E. faecium on improving performance and the condition of gut microbiota in broilers8. The current study aimed to evaluate the best in vitro synbiotic combination between E.faecium and four different prebiotics and evaluate the effect of this synbiotic on the growth performance, microbial population, humoral immune response, and morphology of the small intestine of broiler chickens.

Materials and methods

In vitro studies

Prebiotics are used as substrates

In this research, four different oligosaccharides were utilized as prebiotics. The oligosaccharides included mannan oligosaccharide (Bio-Mos®), oligofructose (with an average degree of polymerization of 4) (Orafti®), galactooligosaccharides (GOS) (with an average degree of polymerization of 6), by Friesland Foods Domo of Holland, and xylooligosaccharides (with an average degree of polymerization of 3), by Shandong Longlive Bio-Technology of China.Prebiotics were kept in the recommended conditions by the manufacturing companies until the experiment began.

Probiotic strain

In this experiment, E. faecium (DSM 3530) was employed as the bacterium. The bacteria were identified using two primary tests, namely Gram and catalase staining tests.

Growth curve of E. faecium bacteria

To identify the bacterial growth pattern of E. faecium bacteria, the growth rate of this bacterium was investigated at different times in 30 h. Ninety cc of MRS broth culture medium was prepared and transferred to 30 test tubes (10 test tubes for 10 different times, each with 3 repetitions, and 3 cc samples in each test tube). The environment’s pH was adjusted to 5.8 ± 1 using hydrochloric acid and 1 normal NaOH. The culture medium temperature was maintained at 41 °C9 To create anaerobic conditions, the test tubes were covered with one cc of sterile liquid paraffin10. After autoclaving and sterilization, the media were inoculated with 1.5 × 108 colony-forming units of E. faecium bacteria, previously cultured on MRS agar. To ensure the anaerobic conditions of the culture environment, the tubes were covered with cotton and aluminum foil after inoculation of bacteria. Then, the test tubes were transferred to a shaker incubator at 41 °C. In order to determine the concentration of bacteria, the test tubes were removed from the incubator at regular intervals after inoculation (every 3 h). Under sterile conditions, 1 cc samples were taken from them and transferred to glass cuvettes. Then, the optical absorption of each cuvette at a wavelength of 600 nm was read and recorded using a spectrophotometer (BRITE Technologies, Canada, Model BT 600)11.

Investigating the ability of E. faecium bacteria to consume prebiotics as a carbon source

The study focused on the synbiotic impacts of E. faecium probiotic bacteria on 4 different polymerized prebiotics, namely mananoligosaccharide, oligofructose, GOS, and xyloligosaccharide, and their comparison to their corresponding control strains. Each prebiotic was considered a treatment, and three repetitions were used for each treatment. The pH, temperature, and anaerobic conditions were adjusted as described after the media was prepared. To prevent the reaction of prebiotics with the components of the environment during autoclaving, prebiotics were added to the environment separately and in the form of a 10% aqueous solution under sterile conditions. The treatment was supplemented with the same amount of distilled water without any probiotics. To inoculate the medium with prebiotics, a fresh culture of E. faecium bacteria was prepared 24 h before the experiment and diluted at 2.5 × 108. After completing the preparation of the experimental treatments in the tubes, the medium was inoculated with E. faecium bacteria at the rate of 1% and with a dilution equal to 1.5 × 108. To determine the growth rate of E. faecium bacteria in each of the treatments and to determine optical density, the test tubes were removed from the incubator at regular intervals after inoculation (every 3 h). Under sterile conditions, a 1 cc sample was taken from them. After removing it, it was transferred to glass cuvettes where it was read and recorded by a spectrophotometer at a wavelength of 600 nm11.

Determining the pH of the environment

The pH level of different treatments was investigated to determine and compare the acidity changes caused by the fermentation of different prebiotics by E. faecium bacteria in the culture medium. For this purpose, after measuring the optical absorption of the samples, the pH of different environments was read and recorded by a digital pH meter.

Number of live E. faecium bacteria in the exponential growth phase

To determine and compare the number of live bacteria in the culture medium containing different prebiotics, the counting of bacteria in the exponential growth phase was investigated. For this purpose, different environments were removed from the incubator 12 h after incubation, and 1 cc sample was taken from them under sterile conditions. After preparing the dilution series of the desired sample, the amount of 0.1 cc of the desired sample was spread on MRS solid culture medium. Finally, after 12 to 24 h of incubation at 41 °C, the number of bacteria was counted12.

In vivo studies

Birds, housing, and management

The animal trial was conducted in accordance with the guidelines was approved the Animal Care and Use Review Committee at Gorgan University of Agricultural Sciences and Natural Resources in Gorgan, Iran (Approval No. AK1390M/32/56). All protocols adhered to the ARRIVE guidelines for reporting animal research (https://arriveguidelines.org). The mechanical cervical dislocation method was used for euthanasia, utilizing the Koechner Euthanizing Device, as recommended by the American Veterinary Medical Association. The required number of 400 broiler chickens of the Cobb 500 breed was procured from Tirgan Bandar’s mother hen company. At the time of egg production, the mother hen flock was in the first production cycle and at the age of 25 weeks, and the average initial weight of the chicks was 35 g, according to the hatching claim. The intended chickens were randomly assigned to 4 experimental treatments, each of which included five repetitions, and each repetition included 20 pieces of one-day-old chickens of two sexes. A control ration was prepared without using any growth-promoting antibiotic and anti-coccidiosis drug and based on the recommended requirements of the Cobb 500 breed. The desired additives were added to the control diet to prepare other treatments at suitable levels as follows. Experimental treatments were applied from the first day until 42 days. The combination of experimental treatments was as follows:

  1. 1.

    Control;

  2. 2.

    Control + prebiotic GOS (500 g per ton of feed).

  3. 3.

    Control + E. faecium probiotic (500 g per ton of feed).

  4. 4.

    Control + selected synbiotic.

Management

Before the arrival of chickens, the temperature was raised to 34 ℃. This temperature decreased by 3 ℃ every week until temperature reached 18 °C, and this temperature remained constant until the end. After entering, experimental chickens were nested randomly inside plastic cages (each pen was 220 × 170 cm2). At the beginning of the arrival of the chickens, water containing sugar and vitamin supplements was provided to them. The chicks had ad libitum to food and water from the first day. In the first week of rearing, water, and feed were provided to the chickens manually and in special floor trays for one-day-old chicks. From the second week to the end of the rearing period, the chickens were fed using hanging feeders and waterers whose height from the ground level was changed according to the age of the chickens. The lighting system was also adjusted based on Cobb 500 recommendations.

Diet preparation

Before preparing experimental rations, the amounts of nutrients in corn and soybeans, which comprised most of the experimental rations, were determined by the NIR device in the laboratory of Evonik Iran. Then, according to the amount of nutrients obtained and based on the recommended requirements of the Cobb 500 breed (2003), a basic diet for three initial periods (1 to 10 days old), grower (11 to 22 days old), and finisher (23 to 42 days) prepared and tested additives were added to it based on their degree of purity. To prepare the desired rations in each period, the values of the basic rations were first weighed for all treatments. Then, the desired amounts were mixed by a 500 kg horizontal mixer for 10 min. After mixing the rations, the amounts required for each experimental treatment were prepared then additives were added manually to the basic rations in several steps. Finally, the desired rations were pelleted by a pellet press machine under a controlled conditioner temperature of 70 ± 2 °C, and after cooling and bagging, they were made available to the birds. It should be noted that 2, 3, and 4 dyes were used to prepare the initial, grower, and finisher diet, respectively. Diet components and nutrient composition of experimental diets used are listed in Table 1.

Table 1 Diet components and nutrient composition of starter, grower and finisher basic diets.
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Growth performance

Chickens of different experimental groups were weighed weekly using a digital scale. The body weight increase of birds was also calculated by subtracting the body weight obtained from the initial weight or the weight of the last week of chickens. It should be mentioned that the chickens were starved before weighing and because the digestive system was empty. The feed consumption of experimental units was determined weekly based on the difference between the amount of feed given at the beginning and the amount of feed remaining at the end of the week. The feed conversion ratio of experimental units was also calculated every week by dividing the amount of feed consumed by the experimental unit by the weight gain of the same experimental unit.

Morphology of the small intestine

To determine the effects of using the tested additives on the morphological characteristics of the small intestine, at the age of 27 and 42 days, five birds from each experimental treatment with a body weight close to the average of each experimental unit were selected. After slaughtering these birds, 2 cm of the middle part of their jejunum was separated, and the food contents in them were slowly removed using geyser. After the initial fixation of the tissues in 4% formalin buffer, the tissues were removed from the solution. After making a longitudinal cut with a surgical blade, they were again kept in formalin until staining and future investigations. Then, five-micron-thick slices were prepared from the jejunum and placed on glass slides. The samples were deparaffinized by xylan solution and dehydrated in graded alcohol solutions. Finally, the samples were stained with hematoxylin and eosin, and the height of the villi, the thickness of the villi, and the depth of the crypts were determined using a light microscope equipped with a camera and analyzed by Image Analysis software13.

Counting the bacterial population and pH of the gastrointestinal tract

One chicken piece was randomly selected from each experimental unit to determine the effects of experimental treatments on the pH and the number of microbial populations in the digestive system at the age of 27 days. After slaughter, it was taken to the laboratory. After disinfecting the abdominal surface of the carcass, the samples obtained from the contents of the ileum and cecum were homogenized with 0.9% saline solution at a ratio of 1:9. After diluting the obtained samples, Lactobacillus, coliform, and Escherichia coli were cultured in their specific culture media. The number of colonies was counted depending on the growth rate of the desired bacteria after 24 to 72 h of incubation at 37 °C. The counting results were multiplied by the dilution ratio and converted into logarithmic data. The pH of the ileum and cecum of slaughtered birds was also read and recorded using a digital pH meter.

Determination of antibody titer against Newcastle virus

To determine and compare the antibody titer against Newcastle virus in different experimental treatments, on the 23d day of the experiment, one chicken was selected from each experimental unit whose weight was as close as possible to the average weight of that experimental unit, and 2 ml of blood was collected through the subclavian vein. Immediately after blood collection, the intended chickens were marked with a spray, and 10 days later, blood collection was done again from the same chickens. After transfer to the laboratory, the antibody titer against Newcastle was determined by the samples’ agglutination inhibition (HI) method (European Pharmacopoeia, 2004). In short, the obtained blood samples were centrifuged for 10 min at 15,000 rpm to prepare serum and blood serum was extracted. 25 µl of serum from blood samples were added to 25 µl of phosphate-buffered saline solution of the first dilution of a 96-well plate, and dilution series was prepared with PBS. After dilution, 25 µl of the antigen solution, which was determined using the HA test, or the amount of 4 units of HA was controlled using the back titration test, was added to all the wells except the first well. The desired plate was incubated and placed on a mechanical stirrer for 30 min at room temperature. Then, 25 µl of 1% red blood cell solution were washed three times with PBS solution and added to all the wells, and the desired plate was again placed on a mechanical stirrer for 30 min at room temperature. Finally, the amount of antibody titer was read and recorded by the log2 dilution of the first well in which the patch was formed.

Statistical analysis

This experiment was conducted as a completely randomized design with four treatments and five repetitions. The one-way analysis of variance test of the SAS software package (2003) was used to analyze the data obtained from this experimental stage. A comparison of means was also done with Duncan’s multi-range test at a significance level of 5%.

Results

Growth of E. faecium bacteria in the presence of different prebiotics under anaerobic conditions

The rate of growth E. faecium bacteria in the presence of various prebiotics with different degrees of polymerization under anaerobic conditions is shown in Fig. 1. The lag phase in the treatments containing GOS and xyloligosaccharide lasted about 6 h. The exponential bacterial growth phase started 9 h after inoculation and continued until about 15 h later. Among experimental treatments, the highest bacterial growth rate was observed in synbiotic treatment containing GOS. The maximum growth rate observed in each of the synbiotic treatments, based on optical turbidity measurement at 600 nm wavelength, is shown in Fig. 1. Compared to other treatments, the highest bacterial growth rate was observed in the treatment containing GOS (P < 0.05).

Fig. 1
figure 1

The growth rate of E. faecium in different synbiotic treatments (Control: 1.22; OF: 3.37; MOS: 1.35; GOS: 6.39; XOS: 5.16) based on optical turbidity measurement at 600 nm (OF oligofructose, MOS Mannanoligosaccharide, GOS galactooligosaccharide, XOS xylooligosaccharide). The bars assigned with different letters denote significant difference (P < 0.05). Values are presented as the mean SE.

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Determine the pH of the environment

To determine and compare the acidity changes caused by the fermentation of probiotics in the culture medium by E. faecium bacteria, the pH of different treatments at the end of the incubation period (24 h) was measured (Fig. 2). The results of this experiment showed that the lowest pH was observed in the synbiotic treatment containing GOS, that there was a significant difference between this treatment and the other treatments (P < 0.05) so that the final pH of the medium was around 1.6 ratio units decreased to the initial pH.

Fig. 2
figure 2

Final pH of media supplemented containing E. faecium and different prebiotics (Control: 5.59; OF: 4.69; MOS: 5.1; GOS: 4.1; XOS: 4.6) after 24 h of anaerobic culture (OF oligofructose, MOS Mannanoligosaccharide, GOS galactooligosaccharide, XOS xylooligosaccharide).The bars assigned with different letters denote significant difference (P < 0.05). Values are presented as the mean SE.

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The results obtained from counting the number of live E. faecium bacteria in the culture medium containing different prebiotics during the exponential growth stage are shown in Fig. 3. In the presence of GOS treatment, the highest bacterial growth rate was observed. (P < 0.05).

Fig. 3
figure 3

The number of live E. faecium bacteria in the culture medium containing different prebiotics (Control: 5.38; OF: 7.45; MOS: 5.58; GOS: 8.04; XOS: 7.64) in the exponential growth phase (OF oligofructose, MOS Mannanoligosaccharide, GOS galactooligosaccharide, XOS xylooligosaccharide). The bars assigned with different letters denote significant difference (P < 0.05). Values are presented as the mean SE.

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Growth performance parameters

Body weight gain

The effect of the experimental treatments on the weight gain of the initial and finisher periods and the entire period is presented in Table 2. In the initial period, despite the numerically positive effect of all treatments on the weight gain of chickens, only the synbiotic treatment could significantly affect the weight gain of chickens compared to the control treatment (P < 0.05). In the finisher period, none of the experimental treatments significantly affected the weight gain of chickens. In the entire rearing period, treatments containing, synbiotic, and prebiotic were able to significantly affect weight gain in comparison with the control treatment (P < 0.05). Despite the numerical improvement of weight gain in the treatment containing probiotics, no significant positive effect was observed when using it on the weight gain of broiler chickens during the entire rearing period.

Table 2 Effect of experimental treatments on the weight gain of broiler chickens in different rearing periods (g).
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Feed intake

The effect of experimental treatments on the feed consumption of broiler chickens in the beginning, end and also the whole period is presented in Table 3. Despite the observation of numerical differences between the treatments used at the beginning, end, and the entire period, there were no significant effects on consumption when using these feed additives.

Table 3 Effect of experimental treatments on the feed intake of broiler chickens in different rearing periods (g).
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Feed conversion ratio

The effect of experimental treatments on the feed conversion ratio of broiler chickens in the beginning, end, and the whole period is presented in Table 4. Despite the observation of numerical differences between the treatments used in the beginning, end, and also the entire period, there were no significant effects when using these feed additives on the coefficient diet conversion of broilers was not observed in any of these rearing periods.

Table 4 Effect of experimental treatments on the feed conversion ratio of broiler chickens in different rearing periods.
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Morphology of small intestine

The effect of the experimental treatments on the microscopic structure of the jejunum of broiler chickens on the 27th and 42nd days of the rearing period is presented in Table 5. As the results of this table show, although on the 27th day, there are numerical differences between the treatments used, especially when using the treatment containing synbiotic regarding the length and thickness of the villi as well as the depth of the crypts, nevertheless no significant effects were observed when using feed additives used in this experiment on these parameters. In any case, the results showed that synbiotic treatment significantly increased the ratio of villus length to crypt depth compared to the control treatment. In this case, no significant difference was observed between other treatments.

Table 5 Effect of experimental treatments on broiler chickens’ microscopic structure of the jejunum on days 27 and 42.
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The investigations conducted on the microscopic structure of the jejunum of broiler chickens at 42 days of age showed no significant difference when using any experimental treatments on any investigated parameters.

Bacterial population and pH of the gastrointestinal tract

The effect of the experimental treatments on the microbial population and pH of the digestive system of broiler chickens on the 27th day of the rearing period is presented in Table 6. As the results of this table show, no significant difference was observed between the experimental treatments regarding the total population of bacteria and lactobacilli and coliforms in the ileum and cecum. Although treatments containing synbiotic and prebiotic significantly reduced the population of E. coli compared to the control treatment (P < 0.05). Birds receiving treatments containing probiotic and synbiotic had lower ileum pH than the control treatment. However, no significant difference was observed between experimental treatments regarding cecum pH.

Table 6 Effect of experimental treatments on microbial population and broiler chickens’ pH of ileum and cecum at the age of 27 days.
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Antibody titer against newcastle virus

The effect of experimental treatments on the antibody titer response against Newcastle virus of broilers on days 10 and 20 after the last vaccination is presented in Table 7. Experimental treatments did not show any significant difference compared to the control group. Similarly, no significant effect was observed when using these feed additives on antibody titer response against Newcastle virus 20 days after the last Newcastle vaccination.

Table 7 Effect of experimental treatments on antibody titer response against Newcastle virus in broilers, 10 and 20 days after the last vaccination (ND HI titer log2).
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Discussion

Regarding the findings of synbiotic effects between different prebiotics and probiotics in vitro, there is limited information on the preferred substrate of each probiotic bacteria to choose the optimal synbiotic combination. This research showed that the highest growth rate of the mentioned synbiotic was related to the treatment containing GOS. After this treatment, synbiotic compounds containing xyloligosaccharide and oligofructose had the highest growth rate. Paying attention to the different degrees of polymerization between the prebiotics used in this experiment shows that all three types of these sugars are among prebiotics with a low degree of polymerization. Audisio et al.14, investigated the relationships between E. faecium and simple carbohydrates. They found that this bacterium grows better in brown sugar and molasses environments than in environments containing white sugar and bagasse. In addition, these researchers found that this synbiotic compound can prevent the growth of the pathogenic bacteria Salmonella pleurum in laboratory conditions. In agreement with the results obtained in this experiment, Saminathan et al.11 investigated the growth rate of 11 lactobacillus bacteria on different commercial prebiotics. They found that most probiotics can easily use GOS and xylooligosaccharides as a substrate. In this regard, Abouloifa et al.15 also observed that xylooligosaccharides are preferred for fermentation by probiotic bacteria.

This experiment showed that the rate of growth and decrease in pH of the environment was higher when probiotics were grown in environments containing prebiotics with a low degree of polymerization. The fermentation ability of probiotics may be directly related to their degree of polymerization. This means that prebiotics with a low degree of polymerization are fermented faster than prebiotics with a high degree of polymerization16. The degree of polymerization of 10 is considered a physicochemical barrier, so oligosaccharides lower than 10 are easily soluble in water, allowing bacteria to use these sugars easily. Hoseinifar et al.17 observed that fructans with a degree of polymerization higher than 10 are fermented by fecal bacteria five times slower than oligosaccharides with a degree of polymerization below 10. Therefore, as the results of this experiment GOS, and xyloligosaccharide, which had a lower degree of polymerization, led to a higher growth of E. faecium compared to other treatments.

This study also showed that the number of living E. faecium in treatments containing GOS, xyloligosaccharide, and oligofructose was significantly higher than in other treatments. This difference can be attributed to the growth rate of bacteria in these environments and the subsequent increase in the number of living bacteria in the environment. Although there was a significant difference in the bacterial growth rate in treatments containing GOS, xyloligosaccharide, and oligofructose, no significant difference was observed. In agreement with the results obtained in this experiment, Khosravi et al.8 also showed that despite the significant difference in bacterial growth on different oligosaccharides, there was a significant difference between these prebiotics when counting the amount of live E. faecium bacteria. In this experiment, the absence of a significant difference between these treatments may have been affected by the sampling time for counting live bacteria. Unlike the treatments containing GOS, where the bacteria entered the exponential growth stage 6 h after inoculation, the bacteria in the oligofructose treatment entered this stage about 9 h after inoculation. Considering that the sampling time for counting live bacteria was 12 h after inoculation, at least part of the significant difference between the treatments containing GOS and xyloligosaccharide with the treatment containing oligofructose may be due to the presence of non-viable bacteria in the environment that may have led to an increase in the optical absorption of these treatments in the spectrophotometer.

Prebiotics in synbiotic compounds should be used carefully and based on the support of laboratory studies. In general, according to the in vitro results obtained under the conditions of this experiment, which show the superiority of the synbiotic combination of E.faecium with GOS compared to other synbiotic compounds used as a suitable additive in poultry nutrition is suggested.

This experiment was conducted to compare the effects of using a synbiotic, whose effects have been proven in vitro, on performance, microbial population, humoral immune response, and morphology of the small intestine of broiler chickens. The results of this experiment showed that the treatment containing synbiotic could significantly affect the weight gain of chickens in the initial rearing period compared to the control treatment. These significant effects and the numerical positive effects observed in the final period led to a significant improvement in weight gain during the entire rearing period in birds receiving treatments containing synbiotics and probiotics. A closer examination of the results of the studies conducted on the use of these additives shows that the effectiveness of these compounds in different experiments is extremely different, which can be caused by various factors, such as environmental factors18, the levels used of these additives19 and nutritional factors12. In agreement with the results obtained in this experiment, the results of some other experiments also showed that the positive effects of the simultaneous use of prebiotics and probiotics in the diet are more significant than when these compounds are used separately20,21. Synergistic effects between probiotics and prebiotics can be found in the definition of these compounds. Based on this, synbiotic are a mixture of probiotics and prebiotics, improving shelf life and binding probiotics to the intestinal wall22. Therefore, the positive effects caused by the use of this synbiotic combination compared to the separate use of these compounds can be attributed to the increase in the number of colonies and the viability of E. faecium bacteria present in this combination and the greater ability of this bacterium to create competitive elimination and binding to attributed active positions23,24.

Some reports show that, in general, these additives cannot directly affect feed intake unless they positively affect weight gain and subsequently increase the metabolic needs of the bird that grows more, which can affect feed consumption25,26.

This study showed that using the studied additives at 27 and 43 days led to positive changes in the measured parameters. Synbiotic treatment significantly increased the ratio of villus length to crypt depth compared to the control treatment. These results are similar to some reports27,28. At the age of 42 days, the use of all the experimental treatments led to a decrease in the thickness of the villi compared to the control treatment. The depth of intestinal crypts indicates the turnover rate of cells in this area29. Under normal growth conditions, these cells have a lifespan of 72 h until the age of four days. Decreasing the depth of the crypts indicates a reduction in the need to build these cells and, ultimately, a decrease in energy consumption in favor of bird growth30. Changes in the morphology of the intestinal wall are strongly affected by the disruption of the balance between pathogenic and non-pathogenic microorganisms31. By adhering to the intestinal wall or producing toxic substances (or both), these pathogens lead to an increase in the turnover of intestinal cells and an increase in the depth of the crypts. In addition, increasing the activity of these bacteria may affect the appearance and secretory properties of the small intestine, cause lesions in the intestinal mucosa layer, and increase the thickness of the villi32,33. One of the most important mechanisms of action of probiotics, prebiotics, or synbiotic in improving the morphological characteristics of the intestine is due to their direct effect on reducing the population of pathogenic bacteria related to the intestinal wall. Therefore, by examining the results obtained in this experiment regarding the microbial population, it can be seen that the use of these additives, especially synbiotics, by reducing the population of coliforms and E. coli, could improve by increasing morphological structure of the intestine and possibly reducing the costs of maintaining the intestine, ultimately provide more nutrients to the bird for production purposes and lead to an improvement in the weight gain of the birds.

Synbiotics can be effective even against Gram-negative bacteria such as E. coli and Salmonella by performing a competitive elimination mechanism34,35. On the other hand, as the results of this experiment also showed, compounds such as synbiotics can reduce the population of Gram-negative bacteria by penetrating the cell membrane by reducing the pH of the environment due to the production and release of volatile fatty acids36,37.

One of the most important mechanisms of action of probiotics and prebiotics is related to their role in stimulating the bird’s immune system. The digestive tract is the site of confrontation between bacteria, antigens, and the bird’s immune system38, and the lymphoid tissues of the cell wall of the digestive tract play a very important role in preparing the reservoir of immune cells to deal with Pathogenic bacteria play a role39. It seems that the effects created when using probiotics, prebiotics, or synbiotics on the immune system are due to the cumulative effects caused by the increase of beneficial bacteria and their products on the responses related to selective receptors of lymphatic tissues obtained from the cell wall of intestinal tissues36. This shows that these compounds can be more effective in conditions of high environmental pollution, bacterial challenges of birds, and lack of effective vaccination programs. This experiment showed that none of the experimental treatments could significantly differ from the control treatment regarding antibody titer against Newcastle virus in 10 and 20 days after vaccination. In agreement with these results, Sadeghi et al.40 reported that the response of birds to antibody titer against Newcastle virus when using probiotics is directly affected by the bird’s microbial population. By comparing the titer of challenged birds with Salmonella and healthy birds, these researchers found that using probiotics only in challenged birds leads to a significant increase in antibody titer.

Conclusion

The present study showed more beneficial effects of GOS on E. faecium growth in an in vitro study, among other investigated prebiotics. The beneficial effects were also observed in vivo, where combined administration of GOS and E. faecium as synbiotic positively affected performance and gut health, especially in the first weeks of production. However, determining the selected synbiotic effects on challenged broiler chickens is a point of interest that merits further research.