**3. Mechanisms of action of prebiotics in poultry**

There are many mechanisms and functions of prebiotics which have been associated with the poultry GIT microbiota, and it seems that there are several bacteria involved in their use; but there is evidence that other microbiota-independent

*Prebiotics and Probiotics - Potential Benefits in Nutrition and Health*

resulting in their total ban as growth-promoting agents [6, 7].

chickens, which has resulted in huge profits for poultry producers [16].

nutrients, herbs, spices, essential oils, plant components, and vaccines [15].

use of antibiotics as growth promoters in the poultry industry.

**2. Types of prebiotics used in the poultry industry**

Among all the available alternatives, prebiotics have proven to be promising alternatives for the poultry industry because they are able to pass through the digestive tract, which facilitates and supports the symbiotic relationship between the host and gastrointestinal tract (GIT) microbiota and results in health benefits for the birds [22–24]. Thus, this chapter exposes the use of prebiotics as feed additives in poultry, with emphasis on their beneficial effects on the microbiota composition, their ability to control pathogenic infections, positive changes in intestinal morphology, improved productive parameters, and immunomodulatory effects as possible mechanisms of action, which make them potential alternatives to avoid the

It is difficult to describe in a few words what a prebiotic is; nevertheless, all definitions agree that these compounds, when administered as feed ingredients, are

Although it is a fact that the reduction or complete abolition in the use of antibiotics for poultry production would have a positive effect in the control of AMR and public health, this would also have negative economic repercussions, since production costs and, consequently, the prices of the final products, as well as the international trade of poultry products, would be affected [17, 18]. Furthermore, the antibioticfree production of poultry could imply public health problems caused by foodborne pathogens such as e *C. perfringens*, *E. coli*, *S. aureus*, *Campylobacter spp*., or *Salmonella spp*. [19–21] while increasing production costs caused by bacterial infections, along with the detriment in the health of the birds and the decrease of the productive parameters. Hence, the ban on the use of antibiotics for poultry production, as well as other increasingly popular trends, such as the growth of the organic products market, has forced poultry producers to find viable alternatives with similar benefits to antibiotics. For that, many specific alternatives have been evaluated and marketed, such as enzymes, prebiotics, probiotics, organic acids, dietary fiber, highly available

in the use of all classes of antimicrobials, with the conditional recommendation not to use those that have been classified as critically important for human medicine, as well as the complete restriction for growth promotion and prevention of infectious diseases that have not yet been clinically diagnosed [5], for which some government regulatory agencies have taken action on the use of antibiotics for animal production,

It could be expected that the total ban on the use of antibiotics as growth promoters will lead to a decrease in the levels of antibiotic resistance [8]. However, we cannot ignore some issues resulting from this ban, such as the increase in the incidence of bacterial infections which would also increase the use of other antibiotics at prophylactic or even therapeutic doses, accelerating the development of AMR in these pathogens and making it a worse scenario [9, 10]. In addition, some farming practices must be implemented to reduce the use of antibiotics in animal production, such as adequate animal vaccination, good hygiene and husbandry practices, higher animal welfare, and improved breeding programs, which implies an increase in production costs, and it is still not enough to completely reduce the risks of infection [11, 12]. Poultry is one of the most commonly exploited species worldwide, and a sector that continues to grow and industrialize in many parts of the world [13], so it was to be expected that a large part of the antibiotics used in animal production was destined to this industry [14, 15]. Antibiotics have been used in poultry production for therapeutic, prophylactic, or growth promotion purposes, especially in broiler

**240**

**Figure 1.**

*Some of the potential mechanisms of action of prebiotics in poultry species, showing how they are metabolized by the host microbiota and have positive effects on immunity, gut health, metabolic activity, and pathogen colonization. Modified and adapted from Refs. [25, 45].*

interactions also exist [30]. Thus, there is no specific mechanism of how prebiotics influence the host animal health or how they can inhibit pathogen establishment, but all their beneficial effects are the result of a complex set of interactions on the host physiology, including their positive effects on intestinal morphology and microbiota balance, the suppressive effects on the enteric and systemic infections by pathogens due to the immune stimulation, the higher nutrient digestibility, and positive metabolic alterations (**Figure 1**), which result in improved productive parameters, higher quality of food products from poultry origin, general welfare of animals, and ultimately decrease in production cost [42, 44].

### **3.1 Intestinal microbiota balance**

In poultry species, as in almost all the others, the microbiota largely determines the intestinal integrity, functionality, and health, which in turn plays a vital role in nutrient digestion and absorption, immune system development, and pathogen exclusion [46]. Since a symbiotic interaction between host and its microbiota is fundamental to poultry health and production, the alteration of the intestinal microbiota when using prebiotic has been one of the most widely investigated mechanisms of action [25, 32, 47]. Nowadays it is well known that prebiotics alter positively the intestinal microbiota in poultry; however, the exact mechanism (s) and type of interaction involved will depend on the structure of the prebiotic and the host species. Among all the microorganisms that make up the intestinal microbiota in poultry, some particular bacteria, such as *Lactobacillus spp.* and *Bifidobacterium spp.*, have positive effects on intestinal physiology and, thus, are beneficial for the host health. These beneficial bacteria, which have been reported to have higher abundance when chickens are fed diets supplemented with prebiotics, can ferment and metabolize prebiotics, selectively stimulating their proliferation and activity [45, 48, 49], which

**243**

to the control group [52].

**Figure 2.**

*Ref. [40].*

with the control group [53].

**3.2 Inhibition of pathogen colonization**

*Use of Prebiotics as an Alternative to Antibiotic Growth Promoters in the Poultry Industry*

can potentially influence the production of short-chain fatty acids (SCFA) and the consequent decrease in intestinal pH, improve the metabolism by increasing digestive enzyme activity and vitamin production and decreasing levels of triglycerides, cholesterol, and odor compounds, and stimulate the immune system that contributes

*Intestinal microbiota modifications in the small intestinal content of broiler chickens at 4 weeks of age. Within a bacterial species (or total), bars with different letters (a–c) are different (P < 0.05, n = 8). Modified from* 

On the other hand, several studies have shown that prebiotics influenced the beneficial intestinal microbiota of broiler chickens while maintaining low levels of potential pathogens in the small intestine and cecal digesta. Addition of FOS as prebiotic to the basal diet (4.0 g/kg) significantly increased the viable count of *Bifidobacterium* and *Lactobacillus* in the small intestinal digesta of male broiler chickens, while the number of *Escherichia coli* was significantly reduced compared

It has been also reported that feeding 0.25% of FOS and 0.05% of MOS to broilers resulted in an increased diversity and population of *Lactobacillus* and decreased populations of *E. coli* and *C. perfringens* in the ileum, as shown in **Figure 2** [40]. In laying hens, dietary supplementation with different levels of inulin linearly reduced coliform bacteria counts by increasing concentrations of this prebiotic, while 2.0% of inulin achieved significantly increased cecal *Bifidobacterium* counts compared

A recent study has shown that 3.5 mg of a GOS mixture delivered in ovo had a bifidogenic effect in adult chickens, since the relative abundance of *Bifidobacterium* communities was higher in four sections of intestinal content (duodenum, jejunum, ileum, and cecum), while the values of *Lactobacillus* abundance resulted to be

The ability of prebiotics in poultry diet to reduce colonization of pathogens results from the combination of several mechanisms occurring in the GIT, from those that are directly related to the selective stimulation of the favorable microbiota to those in which the prebiotics directly affect the pathogens or the host animal in a microbiota-independent manner. To date, it is not possible to define an exact mechanism of prebiotics to reduce pathogenic infections, so more research is

higher in the control group for most of the four sections [32].

required to fully elucidate their exact function and mode of action.

to the inhibitory effects on the growth of pathogenic bacteria [50, 51]**.**

*DOI: http://dx.doi.org/10.5772/intechopen.89053*

*Use of Prebiotics as an Alternative to Antibiotic Growth Promoters in the Poultry Industry DOI: http://dx.doi.org/10.5772/intechopen.89053*

**Figure 2.**

*Prebiotics and Probiotics - Potential Benefits in Nutrition and Health*

interactions also exist [30]. Thus, there is no specific mechanism of how prebiotics influence the host animal health or how they can inhibit pathogen establishment, but all their beneficial effects are the result of a complex set of interactions on the host physiology, including their positive effects on intestinal morphology and microbiota balance, the suppressive effects on the enteric and systemic infections by pathogens due to the immune stimulation, the higher nutrient digestibility, and positive metabolic alterations (**Figure 1**), which result in improved productive parameters, higher quality of food products from poultry origin, general welfare of

*Some of the potential mechanisms of action of prebiotics in poultry species, showing how they are metabolized by the host microbiota and have positive effects on immunity, gut health, metabolic activity, and pathogen* 

In poultry species, as in almost all the others, the microbiota largely determines the intestinal integrity, functionality, and health, which in turn plays a vital role in nutrient digestion and absorption, immune system development, and pathogen exclusion [46]. Since a symbiotic interaction between host and its microbiota is fundamental to poultry health and production, the alteration of the intestinal microbiota when using prebiotic has been one of the most widely investigated mechanisms of action [25, 32, 47]. Nowadays it is well known that prebiotics alter positively the intestinal microbiota in poultry; however, the exact mechanism (s) and type of interaction involved will depend on the structure of the prebiotic and the host species. Among all the microorganisms that make up the intestinal microbiota in poultry, some particular bacteria, such as *Lactobacillus spp.* and *Bifidobacterium spp.*, have positive effects on intestinal physiology and, thus, are beneficial for the host health. These beneficial bacteria, which have been reported to have higher abundance when chickens are fed diets supplemented with prebiotics, can ferment and metabolize prebiotics, selectively stimulating their proliferation and activity [45, 48, 49], which

animals, and ultimately decrease in production cost [42, 44].

**3.1 Intestinal microbiota balance**

*colonization. Modified and adapted from Refs. [25, 45].*

**242**

**Figure 1.**

*Intestinal microbiota modifications in the small intestinal content of broiler chickens at 4 weeks of age. Within a bacterial species (or total), bars with different letters (a–c) are different (P < 0.05, n = 8). Modified from Ref. [40].*

can potentially influence the production of short-chain fatty acids (SCFA) and the consequent decrease in intestinal pH, improve the metabolism by increasing digestive enzyme activity and vitamin production and decreasing levels of triglycerides, cholesterol, and odor compounds, and stimulate the immune system that contributes to the inhibitory effects on the growth of pathogenic bacteria [50, 51]**.**

On the other hand, several studies have shown that prebiotics influenced the beneficial intestinal microbiota of broiler chickens while maintaining low levels of potential pathogens in the small intestine and cecal digesta. Addition of FOS as prebiotic to the basal diet (4.0 g/kg) significantly increased the viable count of *Bifidobacterium* and *Lactobacillus* in the small intestinal digesta of male broiler chickens, while the number of *Escherichia coli* was significantly reduced compared to the control group [52].

It has been also reported that feeding 0.25% of FOS and 0.05% of MOS to broilers resulted in an increased diversity and population of *Lactobacillus* and decreased populations of *E. coli* and *C. perfringens* in the ileum, as shown in **Figure 2** [40]. In laying hens, dietary supplementation with different levels of inulin linearly reduced coliform bacteria counts by increasing concentrations of this prebiotic, while 2.0% of inulin achieved significantly increased cecal *Bifidobacterium* counts compared with the control group [53].

A recent study has shown that 3.5 mg of a GOS mixture delivered in ovo had a bifidogenic effect in adult chickens, since the relative abundance of *Bifidobacterium* communities was higher in four sections of intestinal content (duodenum, jejunum, ileum, and cecum), while the values of *Lactobacillus* abundance resulted to be higher in the control group for most of the four sections [32].

#### **3.2 Inhibition of pathogen colonization**

The ability of prebiotics in poultry diet to reduce colonization of pathogens results from the combination of several mechanisms occurring in the GIT, from those that are directly related to the selective stimulation of the favorable microbiota to those in which the prebiotics directly affect the pathogens or the host animal in a microbiota-independent manner. To date, it is not possible to define an exact mechanism of prebiotics to reduce pathogenic infections, so more research is required to fully elucidate their exact function and mode of action.

Various potential mechanisms have been proposed by which prebiotics can provide resistance to pathogens, one of the main ones being directly related to the beneficial bacteria in the GIT, such as *Lactobacillus* and *Bifidobacterium*, whose selective growth results in an increased concentration of SCFA, especially acetate, propionate, and butyrate, and lactate during primary fermentation process at the ceca [30]. This is mainly because these bacteria secrete several hydrolases, which monogastric animals cannot, hydrolyzing the carbohydrate-based prebiotics through a fermentation process whose metabolic end products not only contribute to the nutrition of poultry, but they have additional beneficial effects [54]. Since SCFA are the principal luminal anions, and they are relatively weak acids, their increased concentration is correlated with a lower intestinal pH, which in turn is associated with a suppression of pathogens by dissipating the proton motive force across the bacterial cell membrane [55], although it has also been reported that SCFA, butyrate specifically, can downregulate expression of invasion genes in *Salmonella* at low doses [56].

For instance, the effect of 14 or 19 days of 10% dietary lactose administration was evaluated in Leghorn chicks, resulting in a significantly increase of acetic, propionic, butyric, and lactic acid concentration in the cecal contents as compared with the control group; additionally, lactose decreased the pH of cecal contents, with the consequent reduction of the total number of chicks with organ cultures that were positives for this pathogen (**Figure 3**) [57].

Another study was conducted to investigate if changes in SCFA could decrease the numbers of *Enterobacteriaceae* in the ceca of broiler chickens during growth; the authors found a significant negative correlation between the log CFU of *Enterobacteriaceae* and the concentration of acetate, and the undissociated form of acetate, propionate, and butyrate, evidencing that SCFA are one of the mechanisms responsible for the decrease in numbers of these bacteria in the ceca of broiler chickens during growth, while they did not affect beneficial GIT bacteria such as *Lactobacillus* [58].

Nevertheless, there are many other mechanisms by which SCFA may be useful to avoid pathogen colonization in the GIT, such as the increased production of mucin by goblet cells that serves as a physical barrier against pathogens and contributes to their lower colonization [59, 60]. The effect of inulin dietary supplementation at different levels on mucin mRNA expression was evaluated at 21 and 42 days in broiler chickens, and it was found that dietary supplementation of this prebiotic at 10 and 15 g/kg enhanced mucin mRNA expression in the jejunum both days [61]. Moreover, depending on the poultry species, the SCFA provide different levels of the of the total metabolic energy requirements, serving as the preferred energy source of colonocytes and stimulating intestinal integrity [54, 62].

#### **Figure 3.**

*Effect of dietary lactose (10%) during 14 and 19 days on cecal organic acid concentrations, pH of cecal contents, and Salmonella enteritidis organ invasion in Leghorn chicks. ( \* ) (\*\*) (\*\*\*) significantly different from controls (P < 0.05) (P < 0.005) (P < 0.001), respectively. Data obtained from Ref. [57].*

**245**

**Figure 4.**

*Use of Prebiotics as an Alternative to Antibiotic Growth Promoters in the Poultry Industry*

On the other hand, the natural antipathogen activity of the intestinal microbiota in poultry has been documented by the Nurmi concept of competitive exclusion, also known as "bacterial antagonism" or "bacterial interference," through which beneficial microorganisms compete with potentially pathogenic bacteria for limiting nutrients and attachment sites on the mucosa, or even by the production of bacteriocins like lactocin, helveticin, curvacin, nisin, or bifidocin, which may be destructive to various Gram-positive or Gram-negative intestinal pathogens, particularly *Salmonella*, *Campylobacter*, and *E. coli* [47, 63, 64]. It has been demonstrated that competitive exclusion is potentiated with prebiotics, since they promote growth of beneficial bacteria which are ubiquitous in the host animal and are

In a study carried out in broiler chicks, the effect of treatments with dietary 7%

nization by *Salmonella typhimurium* (ST) after 10 and 15 days at different inoculum doses, was evaluated. The authors report that treatment with anaerobes without the addition of lactose did not effectively control cecal colonization of ST, while chickens treated with the combination of anaerobic organisms and lactose were resistant to cecal colonization by this pathogen, concluding that oral administration of only total anaerobes did not function well as competitive exclusion cultures [65]. In another similar study, the inhibitory effect of competitive exclusion and 0.1% concentration of FOS, singly and in combination, on *Salmonella enteritidis* SE colonization of chicks was investigated. Chicks received this pathogen at 7 or 21 days, and then birds from each group were slaughtered at 1, 7, and 14 days after for count of SE in cecal contents. Additionally, quantification of the major cecal microbiota was performed. Results from this study demonstrated the efficacy of CE on chicks 7 days post inoculation with SE, but this efficacy was not clearly demonstrated 21 days post inoculation, indicating that the efficacy of CE to reduce susceptibility to SE colonization is higher on young chicks, while FOS offered protection to chickens particularly in 21-day-old chicks (**Figure 4**). Nevertheless, when FOS was given in combination with a CE treatment, both in the 7- and 21-day-old chicks, a reduction in the number of SE per g of ceca was observed, so that low doses of FOS in the diet of chickens with a CE treatment may result in reduced susceptibility to *Salmonella* colonization. Regarding the intestinal microbiota, few changes in *Bifidobacterium*, *Bacteroides*, and *Lactobacillus* in the cecal contents of treated groups were observed compared with the control group, both 7- and 21-day-old chicks, although when chickens were fed FOS for long times, *Bifidobacterium* and/or *Lactobacillus* of the

*Effect of competitive exclusion (CE) and FOS (0.1%) on recovery of Salmonella enteritidis from cecal contents of chicks infected at 7 and 21 days. Numbers in parentheses indicate the number of birds positive for SE/birds examined. The right graph shows the effect of these treatments on the major bacterial population of cecal* 

of anaerobic organisms, alone or in combination, on cecal colo-

*DOI: http://dx.doi.org/10.5772/intechopen.89053*

capable to survive in GIT conditions.

intestinal flora may increase [39].

*microbiota. Data obtained from Ref. [39].*

lactose and 6.3 × 106

#### *Use of Prebiotics as an Alternative to Antibiotic Growth Promoters in the Poultry Industry DOI: http://dx.doi.org/10.5772/intechopen.89053*

On the other hand, the natural antipathogen activity of the intestinal microbiota in poultry has been documented by the Nurmi concept of competitive exclusion, also known as "bacterial antagonism" or "bacterial interference," through which beneficial microorganisms compete with potentially pathogenic bacteria for limiting nutrients and attachment sites on the mucosa, or even by the production of bacteriocins like lactocin, helveticin, curvacin, nisin, or bifidocin, which may be destructive to various Gram-positive or Gram-negative intestinal pathogens, particularly *Salmonella*, *Campylobacter*, and *E. coli* [47, 63, 64]. It has been demonstrated that competitive exclusion is potentiated with prebiotics, since they promote growth of beneficial bacteria which are ubiquitous in the host animal and are capable to survive in GIT conditions.

In a study carried out in broiler chicks, the effect of treatments with dietary 7% lactose and 6.3 × 106 of anaerobic organisms, alone or in combination, on cecal colonization by *Salmonella typhimurium* (ST) after 10 and 15 days at different inoculum doses, was evaluated. The authors report that treatment with anaerobes without the addition of lactose did not effectively control cecal colonization of ST, while chickens treated with the combination of anaerobic organisms and lactose were resistant to cecal colonization by this pathogen, concluding that oral administration of only total anaerobes did not function well as competitive exclusion cultures [65].

In another similar study, the inhibitory effect of competitive exclusion and 0.1% concentration of FOS, singly and in combination, on *Salmonella enteritidis* SE colonization of chicks was investigated. Chicks received this pathogen at 7 or 21 days, and then birds from each group were slaughtered at 1, 7, and 14 days after for count of SE in cecal contents. Additionally, quantification of the major cecal microbiota was performed. Results from this study demonstrated the efficacy of CE on chicks 7 days post inoculation with SE, but this efficacy was not clearly demonstrated 21 days post inoculation, indicating that the efficacy of CE to reduce susceptibility to SE colonization is higher on young chicks, while FOS offered protection to chickens particularly in 21-day-old chicks (**Figure 4**). Nevertheless, when FOS was given in combination with a CE treatment, both in the 7- and 21-day-old chicks, a reduction in the number of SE per g of ceca was observed, so that low doses of FOS in the diet of chickens with a CE treatment may result in reduced susceptibility to *Salmonella* colonization. Regarding the intestinal microbiota, few changes in *Bifidobacterium*, *Bacteroides*, and *Lactobacillus* in the cecal contents of treated groups were observed compared with the control group, both 7- and 21-day-old chicks, although when chickens were fed FOS for long times, *Bifidobacterium* and/or *Lactobacillus* of the intestinal flora may increase [39].

#### **Figure 4.**

*Prebiotics and Probiotics - Potential Benefits in Nutrition and Health*

Various potential mechanisms have been proposed by which prebiotics can provide resistance to pathogens, one of the main ones being directly related to the beneficial bacteria in the GIT, such as *Lactobacillus* and *Bifidobacterium*, whose selective growth results in an increased concentration of SCFA, especially acetate, propionate, and butyrate, and lactate during primary fermentation process at the ceca [30]. This is mainly because these bacteria secrete several hydrolases, which monogastric animals cannot, hydrolyzing the carbohydrate-based prebiotics through a fermentation process whose metabolic end products not only contribute to the nutrition of poultry, but they have additional beneficial effects [54]. Since SCFA are the principal luminal anions, and they are relatively weak acids, their increased concentration is correlated with a lower intestinal pH, which in turn is associated with a suppression of pathogens by dissipating the proton motive force across the bacterial cell membrane [55], although it has also been reported that SCFA, butyrate specifically, can

downregulate expression of invasion genes in *Salmonella* at low doses [56].

that were positives for this pathogen (**Figure 3**) [57].

did not affect beneficial GIT bacteria such as *Lactobacillus* [58].

For instance, the effect of 14 or 19 days of 10% dietary lactose administration was evaluated in Leghorn chicks, resulting in a significantly increase of acetic, propionic, butyric, and lactic acid concentration in the cecal contents as compared with the control group; additionally, lactose decreased the pH of cecal contents, with the consequent reduction of the total number of chicks with organ cultures

Another study was conducted to investigate if changes in SCFA could decrease the numbers of *Enterobacteriaceae* in the ceca of broiler chickens during growth; the authors found a significant negative correlation between the log CFU of *Enterobacteriaceae* and the concentration of acetate, and the undissociated form of acetate, propionate, and butyrate, evidencing that SCFA are one of the mechanisms responsible for the decrease in numbers of these bacteria in the ceca of broiler chickens during growth, while they

Nevertheless, there are many other mechanisms by which SCFA may be useful to avoid pathogen colonization in the GIT, such as the increased production of mucin by goblet cells that serves as a physical barrier against pathogens and contributes to their lower colonization [59, 60]. The effect of inulin dietary supplementation at different levels on mucin mRNA expression was evaluated at 21 and 42 days in broiler chickens, and it was found that dietary supplementation of this prebiotic at 10 and 15 g/kg enhanced mucin mRNA expression in the jejunum both days [61]. Moreover, depending on the poultry species, the SCFA provide different levels of the of the total metabolic energy requirements, serving as the preferred energy source of colonocytes and stimulating intestinal integrity

*Effect of dietary lactose (10%) during 14 and 19 days on cecal organic acid concentrations, pH of cecal contents,* 

*\* ) (\*\*) (\*\*\*)*

 *significantly different from controls* 

*and Salmonella enteritidis organ invasion in Leghorn chicks. (*

*(P < 0.05) (P < 0.005) (P < 0.001), respectively. Data obtained from Ref. [57].*

**244**

**Figure 3.**

[54, 62].

*Effect of competitive exclusion (CE) and FOS (0.1%) on recovery of Salmonella enteritidis from cecal contents of chicks infected at 7 and 21 days. Numbers in parentheses indicate the number of birds positive for SE/birds examined. The right graph shows the effect of these treatments on the major bacterial population of cecal microbiota. Data obtained from Ref. [39].*

Some pathogenic bacteria, such as *Salmonella spp., E. coli*, or *Vibrio cholerae*, have mannose-specific lectins (Type 1 fimbriae) on their surface, which recognize glycoprotein receptors rich in mannose on the intestinal cells of the host animal and are key to initiate attachment and colonization [45, 48]. Prebiotics, specifically MOS, can also reduce pathogen colonization by their direct union to the pathogen lectins, avoiding its attachment to the intestinal epithelial cells and, thus, passing through the GIT without colonizing.

This mechanism has been also corroborated in poultry species, both in vitro and in vivo, in two independent studies. In the first study, the in vitro effect of D-mannose, galactose, methyl-α-D-mannoside, and arabinose, on the adherence of *Salmonella typhimurium* to epithelial cells of the small intestine from 1-day-old chicks, was investigated. Authors showed that the small intestine of the chicken has receptors for bacteria with Type 1 fimbriae, and those fimbriae-positive strains of ST adhered significantly better than fimbriae-negative strains. They reported that adherence of ST to chicken small intestinal cells was inhibited more than 90% by methyl-α-D-mannoside and D-mannose and to a lesser extent by arabinose and galactose through the mechanism of blocking [66].

In the other study, the same effect of mannose was demonstrated in vivo. For that, 1-day-old broiler chickens were fed normal drinking water or drinking water supplemented with mannose (2.5% w/v) for 10 days. On day 3, birds were challenged orally with *S. typhimurium* (108 CFU), and then the cecal contents were examined on day 10. Results corroborated the blocking action of D-mannose, which could reduce the percentage of chickens colonized by ST from 78 to 28%, 82 to 21%, and 93 to 43%, in three trials [67].

In a more recent study, the ability of MOS from yeast cell walls to decrease the concentrations of enteric pathogens that express Type 1 fimbriae in poultry was evaluated. In the first part of this work, the ability of different enteric pathogens and coliforms to adhere to the MOS was measured in vitro, evaluating qualitatively if agglutination was modified with the presence of fructose, galactose, glucose, and mannose. Results of the agglutination test showed that 5 of 7 strains of *Escherichia coli* and 7 of 10 strains of *Salmonella typhimurium* and *S. enteritidis* agglutinated MOS. Other strains like *S. montevideo*, *S. give*, *S. kedougou*, and *S. dublin* also caused agglutination of MOS, but strains of *S. choleraesuis*, *S. pullorum*, and *Campylobacter* did not lead to agglutination. Nevertheless, agglutination of these Gram-negative bacteria could be inhibited by mannose and fructose, although it took much more fructose to observe the inhibitory responses than mannose. Authors reported that MOS had reduced cecal *S. typhimurium* concentrations by about 25-fold compared to the control group; concentrations of coliforms also tended to be lower when MOS was added to the feed, while concentrations of *lactobacilli*, *enterococci*, and anaerobic bacteria were not affected by treatment; concentration mean values are shown in the upper left graph of **Figure 5**. This tendency to reduce the salmonella concentration was observed during the time after the challenge with the pathogen, as shown in the upper right graph of **Figure 5**. The last part of the study consisted in a similar challenge using *S. dublin*, in which the percentage of prevalence this pathogen was lower in the MOS-treated groups (55.7%) than in the control group (89.8%), while concentrations of the other bacterial populations were not different. Since no changes in cecal parameters were observed with MOS addition, such as a major shift in bacterial populations or changes in pH or SCFA concentrations, which are known to affect salmonella, together with the in vitro agglutination results, authors conclude that adsorption of salmonellae by MOS could be a possible mode of action by which adhesion of these pathogens to the wall is avoided [68].

**247**

*Use of Prebiotics as an Alternative to Antibiotic Growth Promoters in the Poultry Industry*

Another proposed mechanism for health benefits of prebiotics is the improved intestinal morphological structure; several prebiotics have proven their capacity to modify positively intestinal morphology, both on macroscopic (intestinal length) and microscopic (size and density of villi and microvilli and crypt depth) structures of different sections of the intestine in poultry species [52, 69, 70]. Furthermore, an increased number of goblet cells of the intestinal villi have been reported after dietary administration of prebiotics; these specialized cells are responsible for secreting glycoprotein compounds, mainly mucins, which bind pathogenic microorganisms and reduce their adherence to the intestinal mucosa [71]. These morphological changes lead to a higher efficiency of nutrient absorption, since well-developed and functional enterocytes have been associated with increased absorptive area of the intestine [72] but also with an increased activity of the intestinal brush border enzymes and the nutrient

*letters (a, b) are different (P < 0.05, n = 6). Data were obtained from Ref. [68].*

*Upper graphs: Effect of dietary added MOS on concentrations of different bacterial populations (left) and concentration of Salmonella typhimurium at different times after challenge in the ceca of chicks (right). Lower graphs show different bacterial populations (left) in the ceca of chicks challenged with S. dublin and (right) the percentage of birds from which S. dublin was recovered. Within bacterial populations, bars with different* 

In turkeys, the dietary addition of two doses of a product based on MOS and β-glucans (1 and 2 lb./ton) on gastrointestinal tract development was evaluated through the measurement of ileal, jejunal, and duodenal morphology of turkey poults at 7 and 21 days of age. Data derived from this study suggest that feed supplemented with MOS and β-glucans could accelerate GIT maturation in turkey poults and was more pronounced in the ileum than in other portions of the small intestine. Ileum villus height, surface area, lamina propria thickness, and crypt depth were enhanced with the prebiotic treatment both on day 7 and 21, in a dose-dependent manner for many of the parameters evaluated, as it can be observed in **Figure 6**. In the jejunum results were consistently higher only for the highest dose of treatment (2 lb./ton) compared with the control group on both days, while in the duodenum results were better for the highest dose on day 7, although intestinal morphology of this intestinal section was not different on day 21. Furthermore, density of neutral, sialomucin, and sulfomucin goblet cells that were taken per intestinal section was also evaluated, showing a very similar tendency than the enteric morphometric

*DOI: http://dx.doi.org/10.5772/intechopen.89053*

**3.3 Intestinal morphology**

**Figure 5.**

transport systems [70, 73].

*Use of Prebiotics as an Alternative to Antibiotic Growth Promoters in the Poultry Industry DOI: http://dx.doi.org/10.5772/intechopen.89053*

#### **Figure 5.**

*Prebiotics and Probiotics - Potential Benefits in Nutrition and Health*

through the GIT without colonizing.

galactose through the mechanism of blocking [66].

which adhesion of these pathogens to the wall is avoided [68].

lenged orally with *S. typhimurium* (108

and 93 to 43%, in three trials [67].

Some pathogenic bacteria, such as *Salmonella spp., E. coli*, or *Vibrio cholerae*, have mannose-specific lectins (Type 1 fimbriae) on their surface, which recognize glycoprotein receptors rich in mannose on the intestinal cells of the host animal and are key to initiate attachment and colonization [45, 48]. Prebiotics, specifically MOS, can also reduce pathogen colonization by their direct union to the pathogen lectins, avoiding its attachment to the intestinal epithelial cells and, thus, passing

This mechanism has been also corroborated in poultry species, both in vitro and in vivo, in two independent studies. In the first study, the in vitro effect of D-mannose, galactose, methyl-α-D-mannoside, and arabinose, on the adherence of *Salmonella typhimurium* to epithelial cells of the small intestine from 1-day-old chicks, was investigated. Authors showed that the small intestine of the chicken has receptors for bacteria with Type 1 fimbriae, and those fimbriae-positive strains of ST adhered significantly better than fimbriae-negative strains. They reported that adherence of ST to chicken small intestinal cells was inhibited more than 90% by methyl-α-D-mannoside and D-mannose and to a lesser extent by arabinose and

In the other study, the same effect of mannose was demonstrated in vivo. For that, 1-day-old broiler chickens were fed normal drinking water or drinking water supplemented with mannose (2.5% w/v) for 10 days. On day 3, birds were chal-

examined on day 10. Results corroborated the blocking action of D-mannose, which could reduce the percentage of chickens colonized by ST from 78 to 28%, 82 to 21%,

In a more recent study, the ability of MOS from yeast cell walls to decrease the concentrations of enteric pathogens that express Type 1 fimbriae in poultry was evaluated. In the first part of this work, the ability of different enteric pathogens and coliforms to adhere to the MOS was measured in vitro, evaluating qualitatively if agglutination was modified with the presence of fructose, galactose, glucose, and mannose. Results of the agglutination test showed that 5 of 7 strains of *Escherichia coli* and 7 of 10 strains of *Salmonella typhimurium* and *S. enteritidis* agglutinated MOS. Other strains like *S. montevideo*, *S. give*, *S. kedougou*, and *S. dublin* also caused agglutination of MOS, but strains of *S. choleraesuis*, *S. pullorum*, and *Campylobacter* did not lead to agglutination. Nevertheless, agglutination of these Gram-negative bacteria could be inhibited by mannose and fructose, although it took much more fructose to observe the inhibitory responses than mannose. Authors reported that MOS had reduced cecal *S. typhimurium* concentrations by about 25-fold compared to the control group; concentrations of coliforms also tended to be lower when MOS was added to the feed, while concentrations of *lactobacilli*, *enterococci*, and anaerobic bacteria were not affected by treatment; concentration mean values are shown in the upper left graph of **Figure 5**. This tendency to reduce the salmonella concentration was observed during the time after the challenge with the pathogen, as shown in the upper right graph of **Figure 5**. The last part of the study consisted in a similar challenge using *S. dublin*, in which the percentage of prevalence this pathogen was lower in the MOS-treated groups (55.7%) than in the control group (89.8%), while concentrations of the other bacterial populations were not different. Since no changes in cecal parameters were observed with MOS addition, such as a major shift in bacterial populations or changes in pH or SCFA concentrations, which are known to affect salmonella, together with the in vitro agglutination results, authors conclude that adsorption of salmonellae by MOS could be a possible mode of action by

CFU), and then the cecal contents were

**246**

*Upper graphs: Effect of dietary added MOS on concentrations of different bacterial populations (left) and concentration of Salmonella typhimurium at different times after challenge in the ceca of chicks (right). Lower graphs show different bacterial populations (left) in the ceca of chicks challenged with S. dublin and (right) the percentage of birds from which S. dublin was recovered. Within bacterial populations, bars with different letters (a, b) are different (P < 0.05, n = 6). Data were obtained from Ref. [68].*

#### **3.3 Intestinal morphology**

Another proposed mechanism for health benefits of prebiotics is the improved intestinal morphological structure; several prebiotics have proven their capacity to modify positively intestinal morphology, both on macroscopic (intestinal length) and microscopic (size and density of villi and microvilli and crypt depth) structures of different sections of the intestine in poultry species [52, 69, 70]. Furthermore, an increased number of goblet cells of the intestinal villi have been reported after dietary administration of prebiotics; these specialized cells are responsible for secreting glycoprotein compounds, mainly mucins, which bind pathogenic microorganisms and reduce their adherence to the intestinal mucosa [71]. These morphological changes lead to a higher efficiency of nutrient absorption, since well-developed and functional enterocytes have been associated with increased absorptive area of the intestine [72] but also with an increased activity of the intestinal brush border enzymes and the nutrient transport systems [70, 73].

In turkeys, the dietary addition of two doses of a product based on MOS and β-glucans (1 and 2 lb./ton) on gastrointestinal tract development was evaluated through the measurement of ileal, jejunal, and duodenal morphology of turkey poults at 7 and 21 days of age. Data derived from this study suggest that feed supplemented with MOS and β-glucans could accelerate GIT maturation in turkey poults and was more pronounced in the ileum than in other portions of the small intestine. Ileum villus height, surface area, lamina propria thickness, and crypt depth were enhanced with the prebiotic treatment both on day 7 and 21, in a dose-dependent manner for many of the parameters evaluated, as it can be observed in **Figure 6**. In the jejunum results were consistently higher only for the highest dose of treatment (2 lb./ton) compared with the control group on both days, while in the duodenum results were better for the highest dose on day 7, although intestinal morphology of this intestinal section was not different on day 21. Furthermore, density of neutral, sialomucin, and sulfomucin goblet cells that were taken per intestinal section was also evaluated, showing a very similar tendency than the enteric morphometric

#### **Figure 6.**

*Effect of MOS dietary addition at two different doses on the ileal, jejunal, and duodenal morphology of Turkey poults at 7 and 21 days of age. Within treatments, bars with different letters (a–c) are different (P < 0.05, N = 9 birds, n = 20 measurements/bird). Data were obtained from Ref. [74].*

evaluation, providing evidence of the immunostimulatory effects of this MOS- and β-glucan-based additive, because the numbers of neutral, sialomucin, and sulfomucin goblet cells in the GIT were increased in supplemented poults [74].

Studies have also been conducted whose results demonstrate the beneficial effect of prebiotics on changes at the macroscopic level. A study to evaluate and compare the effectiveness of adding inulin (1%) and oligofructose (1%) to the feed of broiler chickens was conducted, being one of the objectives to evaluate the intestinal length considering the influence of the bird sex. The experiment the experiment lasted 6 weeks, during which the productive parameters were also evaluated. Results from this study suggest that the longer the intestinal length, the better in nutrient absorption which resulted in a heavier body weight, showing correlation coefficients between intestinal length and body weight of 0.68 and 0.74 for the male and female birds, respectively, regardless of the treatments. Oligofructose-treated birds resulted to have a longer intestinal length, especially for the females, although inulin-fed birds also had a longer small intestine than control birds. There were no visible differences in villi density among the males, regardless of the treatments, while for females, the villi from inulin- and oligofructose-treated birds appeared to be denser than those of the controls [69].

#### **3.4 Productive performance**

Undoubtedly, one of the main objectives of the use of food additives in the poultry industry is the improvement of productive performance, a major indicator of poultry well-being that is directly tied to efficiency of nutrients utilization and, thus, to the profitability of production. In fact, replacement of antibiotics as growth promoters with prebiotics to observe improvements in poultry performance is the major reason for the researches [28]. As mentioned above, there is no exact mechanism of action for beneficial effects of prebiotics, so that stimulation of poultry performance results from the very complex interactions of all mechanisms previously described, for instance, by decreasing pathogen colonization, since it

**249**

*Use of Prebiotics as an Alternative to Antibiotic Growth Promoters in the Poultry Industry*

has been described that pathogens depress performance by interfering with nutrient digestion, absorption, and utilization; impairment of normal cellular function; negative impact on enzyme activity, epithelial integrity, and function; and diversion of energy for growth to immune response purposes [75]. Prebiotics can potentially stimulate growth performance through increased SCFA production in poultry, mostly acetate, propionate, and butyrate, since they are directly absorbed in the intestine and used as an energy source in tissues and besides that stimulate a higher metabolic activity in the intestine [76, 77]. SCFA may also serve as a potent regulator of insulin homeostasis in the chicken and carbohydrate metabolism, which stimulate the metabolic activity of striated muscle cells, possibly also having an influence on muscle protein synthesis and, thus, growth performance [78]. Beneficial effects of prebiotic on nutrient digestibility of poultry have been also reported, such as improved digestibility of crude protein, fat, dry matter, energy, and minerals [79, 80]. These results have been attributed to an increase in the beneficial microbiota, such as *Lactobacillus*, changes in the intestinal mucosal structure, and improved intestinal health, which result from the morphological changes in the intestine that lead to a higher efficiency of nutrient absorption and a better nutrient

Other prebiotic effects that might influence productive performance of poultry species are alterations on lipid metabolism and mineral absorption [83, 84]. Studies have demonstrated that prebiotic supplements have a positive effect on the mineral metabolism of Ca, P, Zn, Cu, and Fe [85–88], whose intake is influenced by factors such as the lower luminal pH that increases their solubility promotion and thus their passive absorption, changes in the intestinal mucosa and increased absorption surface area, elevated expression of Ca-binding proteins, release of bone-modulating factors, phytate degradation by probiotic bacteria enzymes, and improved overall intestinal health [89, 90]. On the other hand, although no exact mechanisms have been reported for the alteration on lipid metabolism caused by prebiotics, it has been demonstrated that intestinal microbiota play a role in maintaining lipid metabolism [91], so that the increase in bacterial numbers or a change in the composition of the intestinal microbiota might be related to the lipidic alterations. Studies have shown that prebiotics have a positive effect on lipid metabolism in poultry species, such as hypocholesterolemic effect both in serum and eggs, which has been attributed to many reasons. The enhanced production of SCFA results in inhibition of cholesterol biosynthesis in the liver, due to inhibition of the incorporation of colonic acetate into plasma lipids [92]. Another mechanism through which prebiotics may exert hypocholesterolemic effect is via bile acids, since they enter the small intestine and are absorbed and directed to the liver; however, during reabsorption, conjugated bile acids are exposed to intestinal microflora that hydrolyze conjugated bile acids, making cholesterol unavailable for absorption into the circulation [53]. Although it has not been evaluated in poultry, other studies have also suggested that prebiotics may modify gene expression of lipogenic enzymes, with reduced concentration of plasma phospholipids, triacylglycerols, and lipoproteins [93–96]. However, reports of prebiotics on the performance of poultry have been very variable, and often contradictory, as their effectiveness is strongly dependent on the type of prebiotic and the source, dose used, time of consumption, type of diet and interactions with other feed additives, administration route, animal characteristics, hygiene, husbandry conditions, and environmental stress [28, 50]. In a study carried out in White Leghorn hens, the performance parameters were measured to test two prebiotic treatments consumed for 4 weeks, oligofructose (1% w/w) and inulin (1% w/w), during the later part of the first laying cycle. Egg production, cumulative egg weight per bird, and average egg weight for each treatment were calculated weekly. Besides, body weight change, feed consumption, and

*DOI: http://dx.doi.org/10.5772/intechopen.89053*

transport system, as discussed above [70, 81, 82].

#### *Use of Prebiotics as an Alternative to Antibiotic Growth Promoters in the Poultry Industry DOI: http://dx.doi.org/10.5772/intechopen.89053*

has been described that pathogens depress performance by interfering with nutrient digestion, absorption, and utilization; impairment of normal cellular function; negative impact on enzyme activity, epithelial integrity, and function; and diversion of energy for growth to immune response purposes [75]. Prebiotics can potentially stimulate growth performance through increased SCFA production in poultry, mostly acetate, propionate, and butyrate, since they are directly absorbed in the intestine and used as an energy source in tissues and besides that stimulate a higher metabolic activity in the intestine [76, 77]. SCFA may also serve as a potent regulator of insulin homeostasis in the chicken and carbohydrate metabolism, which stimulate the metabolic activity of striated muscle cells, possibly also having an influence on muscle protein synthesis and, thus, growth performance [78]. Beneficial effects of prebiotic on nutrient digestibility of poultry have been also reported, such as improved digestibility of crude protein, fat, dry matter, energy, and minerals [79, 80]. These results have been attributed to an increase in the beneficial microbiota, such as *Lactobacillus*, changes in the intestinal mucosal structure, and improved intestinal health, which result from the morphological changes in the intestine that lead to a higher efficiency of nutrient absorption and a better nutrient transport system, as discussed above [70, 81, 82].

Other prebiotic effects that might influence productive performance of poultry species are alterations on lipid metabolism and mineral absorption [83, 84]. Studies have demonstrated that prebiotic supplements have a positive effect on the mineral metabolism of Ca, P, Zn, Cu, and Fe [85–88], whose intake is influenced by factors such as the lower luminal pH that increases their solubility promotion and thus their passive absorption, changes in the intestinal mucosa and increased absorption surface area, elevated expression of Ca-binding proteins, release of bone-modulating factors, phytate degradation by probiotic bacteria enzymes, and improved overall intestinal health [89, 90]. On the other hand, although no exact mechanisms have been reported for the alteration on lipid metabolism caused by prebiotics, it has been demonstrated that intestinal microbiota play a role in maintaining lipid metabolism [91], so that the increase in bacterial numbers or a change in the composition of the intestinal microbiota might be related to the lipidic alterations. Studies have shown that prebiotics have a positive effect on lipid metabolism in poultry species, such as hypocholesterolemic effect both in serum and eggs, which has been attributed to many reasons. The enhanced production of SCFA results in inhibition of cholesterol biosynthesis in the liver, due to inhibition of the incorporation of colonic acetate into plasma lipids [92]. Another mechanism through which prebiotics may exert hypocholesterolemic effect is via bile acids, since they enter the small intestine and are absorbed and directed to the liver; however, during reabsorption, conjugated bile acids are exposed to intestinal microflora that hydrolyze conjugated bile acids, making cholesterol unavailable for absorption into the circulation [53]. Although it has not been evaluated in poultry, other studies have also suggested that prebiotics may modify gene expression of lipogenic enzymes, with reduced concentration of plasma phospholipids, triacylglycerols, and lipoproteins [93–96]. However, reports of prebiotics on the performance of poultry have been very variable, and often contradictory, as their effectiveness is strongly dependent on the type of prebiotic and the source, dose used, time of consumption, type of diet and interactions with other feed additives, administration route, animal characteristics, hygiene, husbandry conditions, and environmental stress [28, 50].

In a study carried out in White Leghorn hens, the performance parameters were measured to test two prebiotic treatments consumed for 4 weeks, oligofructose (1% w/w) and inulin (1% w/w), during the later part of the first laying cycle. Egg production, cumulative egg weight per bird, and average egg weight for each treatment were calculated weekly. Besides, body weight change, feed consumption, and

*Prebiotics and Probiotics - Potential Benefits in Nutrition and Health*

evaluation, providing evidence of the immunostimulatory effects of this MOS- and β-glucan-based additive, because the numbers of neutral, sialomucin, and sulfomu-

*Effect of MOS dietary addition at two different doses on the ileal, jejunal, and duodenal morphology of Turkey poults at 7 and 21 days of age. Within treatments, bars with different letters (a–c) are different (P < 0.05,* 

Undoubtedly, one of the main objectives of the use of food additives in the poultry industry is the improvement of productive performance, a major indicator of poultry well-being that is directly tied to efficiency of nutrients utilization and, thus, to the profitability of production. In fact, replacement of antibiotics as growth promoters with prebiotics to observe improvements in poultry performance is the major reason for the researches [28]. As mentioned above, there is no exact mechanism of action for beneficial effects of prebiotics, so that stimulation of poultry performance results from the very complex interactions of all mechanisms previously described, for instance, by decreasing pathogen colonization, since it

Studies have also been conducted whose results demonstrate the beneficial effect of prebiotics on changes at the macroscopic level. A study to evaluate and compare the effectiveness of adding inulin (1%) and oligofructose (1%) to the feed of broiler chickens was conducted, being one of the objectives to evaluate the intestinal length considering the influence of the bird sex. The experiment the experiment lasted 6 weeks, during which the productive parameters were also evaluated. Results from this study suggest that the longer the intestinal length, the better in nutrient absorption which resulted in a heavier body weight, showing correlation coefficients between intestinal length and body weight of 0.68 and 0.74 for the male and female birds, respectively, regardless of the treatments. Oligofructose-treated birds resulted to have a longer intestinal length, especially for the females, although inulin-fed birds also had a longer small intestine than control birds. There were no visible differences in villi density among the males, regardless of the treatments, while for females, the villi from inulin- and oligofructose-treated birds appeared to

cin goblet cells in the GIT were increased in supplemented poults [74].

*N = 9 birds, n = 20 measurements/bird). Data were obtained from Ref. [74].*

be denser than those of the controls [69].

**3.4 Productive performance**

**248**

**Figure 6.**

feed conversion ratio were also monitored. Results showed that oligofructose and inulin increased weekly egg production by 13.35 and 10.73% and cumulative weekly egg weight per bird by 12.50 and 10.96%, respectively, as compared to the control group. Both prebiotics also improved the feed conversion ratio. Nevertheless, there were no differences in average egg weight, feed consumption, or the percentages of changes in live body weight after 4 weeks, as shown in **Figure 7** [85].

In another study, the effect of MOS at a dosage of 2 g/kg on growth performance and nutrient digestibility of two cereal-based diets (corn or wheat) in broiler chickens was evaluated, over an experimental period of 21 days. For that, body weight, feed intake, and feed conversion ration were measured at week 1 and weeks 2–3. Also, the ileal digestibility of nutrients was evaluated on day 21. Authors reported that dietary addition of MOS did not affect the body weight gain of birds but increased their feed intake during the first 7 days, while the feed conversion ratio also tended to increase with MOS, regardless of the type of cereal-based diet. Contrary, between 7 and 21 days, dietary MOS improved the growth performance of birds given the wheat-based diet compared to that of birds given the corn-based diet. Regarding the ileal digestibility of starch, the addition of MOS improved it and showed a high interaction with the type of cereal, indicating that this positive effect of MOS was more profound for the wheat diet than for the corn diet.

#### **3.5 Enhancement of immune system**

Currently, much research has focused in modulation of the immune system by the use of prebiotics, which results from the interaction of all the mechanisms mentioned above, so that it is not an isolated mechanism. A multitude of mechanisms and functions associated with the immunomodulatory effect of prebiotics have been reported, by the activation of genes and pathways implicated in immune processes [25]. It has been cited for many authors that the use of prebiotics in poultry diets improves bird's immunity through the selective growth of beneficial microbiota, resulting in an increased production of a variety of substances, such as bacteriocins and SCFA, that, in addition to being able to inhibit growth of pathogens, play a role in signaling pathway of immune system [97–99].

#### **Figure 7.**

*Performance parameters as affected by dietary oligofructose and inulin in laying hens. Within treatments, bars with different letters (a, b) are different (P < 0.05, n = 10). Data were obtained from Ref. [85].*

**251**

**Figure 8.**

*Use of Prebiotics as an Alternative to Antibiotic Growth Promoters in the Poultry Industry*

The chicken gut microbiota, especially *Lactobacillus* and *Bifidobacterium*, has also been reported to modulate intestinal gene expression, T cell-mediated immunity, and accelerated intestinal immune system maturation, by influencing the intestinal epithelium to produce antimicrobial peptides and cytokines such as IL-12, IFN-γ, IL-10, IL-1β, and TNF-α; modulating the immune system through enhancement of phagocytosis and proliferation of immune cells such as macrophages and monocytes; enhancing production of IgA, IgM, and IgG, reactive oxygen species, and reactive nitrogen species; and proliferating natural killer cells, CD3, CD4, and CD8 T cells [25, 47, 75, 100]. Some prebiotics have shown to increase the production of secretory IgA in the intestine, which inhibits the attachment and penetration of bacteria in the lumen, increases the production of mucus, and prevents inflamma-

On the other hand, as previously mentioned, prebiotics can inhibit pathogen colonization, decreasing detrimental molecules produced by pathogenic bacteria, which act as exogenous signals called pathogen-associated molecular patterns (PAMPs). These PAMPs can be recognized by pattern recognition receptors (PRR)

*Relative mRNA expression of immune-related (cytokines and host defense peptides) and barrier function (mucin and free fatty acid receptors) genes in different segments of intestinal mucosa in chickens injected in ovo with GOS. Asterisk indicates pair-wise significant differences (P < 0.05, n = 10). Graphs were obtained from Ref. [32].*

*DOI: http://dx.doi.org/10.5772/intechopen.89053*

tion that could cause epithelial tissue damage [40, 42].

*Use of Prebiotics as an Alternative to Antibiotic Growth Promoters in the Poultry Industry DOI: http://dx.doi.org/10.5772/intechopen.89053*

The chicken gut microbiota, especially *Lactobacillus* and *Bifidobacterium*, has also been reported to modulate intestinal gene expression, T cell-mediated immunity, and accelerated intestinal immune system maturation, by influencing the intestinal epithelium to produce antimicrobial peptides and cytokines such as IL-12, IFN-γ, IL-10, IL-1β, and TNF-α; modulating the immune system through enhancement of phagocytosis and proliferation of immune cells such as macrophages and monocytes; enhancing production of IgA, IgM, and IgG, reactive oxygen species, and reactive nitrogen species; and proliferating natural killer cells, CD3, CD4, and CD8 T cells [25, 47, 75, 100]. Some prebiotics have shown to increase the production of secretory IgA in the intestine, which inhibits the attachment and penetration of bacteria in the lumen, increases the production of mucus, and prevents inflammation that could cause epithelial tissue damage [40, 42].

On the other hand, as previously mentioned, prebiotics can inhibit pathogen colonization, decreasing detrimental molecules produced by pathogenic bacteria, which act as exogenous signals called pathogen-associated molecular patterns (PAMPs). These PAMPs can be recognized by pattern recognition receptors (PRR)

#### **Figure 8.**

*Prebiotics and Probiotics - Potential Benefits in Nutrition and Health*

feed conversion ratio were also monitored. Results showed that oligofructose and inulin increased weekly egg production by 13.35 and 10.73% and cumulative weekly egg weight per bird by 12.50 and 10.96%, respectively, as compared to the control group. Both prebiotics also improved the feed conversion ratio. Nevertheless, there were no differences in average egg weight, feed consumption, or the percentages of

In another study, the effect of MOS at a dosage of 2 g/kg on growth performance

changes in live body weight after 4 weeks, as shown in **Figure 7** [85].

of MOS was more profound for the wheat diet than for the corn diet.

gens, play a role in signaling pathway of immune system [97–99].

Currently, much research has focused in modulation of the immune system by the use of prebiotics, which results from the interaction of all the mechanisms mentioned above, so that it is not an isolated mechanism. A multitude of mechanisms and functions associated with the immunomodulatory effect of prebiotics have been reported, by the activation of genes and pathways implicated in immune processes [25]. It has been cited for many authors that the use of prebiotics in poultry diets improves bird's immunity through the selective growth of beneficial microbiota, resulting in an increased production of a variety of substances, such as bacteriocins and SCFA, that, in addition to being able to inhibit growth of patho-

*Performance parameters as affected by dietary oligofructose and inulin in laying hens. Within treatments, bars* 

*with different letters (a, b) are different (P < 0.05, n = 10). Data were obtained from Ref. [85].*

**3.5 Enhancement of immune system**

and nutrient digestibility of two cereal-based diets (corn or wheat) in broiler chickens was evaluated, over an experimental period of 21 days. For that, body weight, feed intake, and feed conversion ration were measured at week 1 and weeks 2–3. Also, the ileal digestibility of nutrients was evaluated on day 21. Authors reported that dietary addition of MOS did not affect the body weight gain of birds but increased their feed intake during the first 7 days, while the feed conversion ratio also tended to increase with MOS, regardless of the type of cereal-based diet. Contrary, between 7 and 21 days, dietary MOS improved the growth performance of birds given the wheat-based diet compared to that of birds given the corn-based diet. Regarding the ileal digestibility of starch, the addition of MOS improved it and showed a high interaction with the type of cereal, indicating that this positive effect

**250**

**Figure 7.**

*Relative mRNA expression of immune-related (cytokines and host defense peptides) and barrier function (mucin and free fatty acid receptors) genes in different segments of intestinal mucosa in chickens injected in ovo with GOS. Asterisk indicates pair-wise significant differences (P < 0.05, n = 10). Graphs were obtained from Ref. [32].*

expressed on the surface of epithelial cells, macrophages, mast cells, and dendritic cells, including toll-like receptors and NOD-like receptors, and once recognized are activated, producing cytokines for the regulation of further innate immune responses [45]. Although little data show direct effects of prebiotics on immune function, some studies have indicated that prebiotics have an improved response to salmonella vaccine, which could be because prebiotics can act as nonpathogenic antigens themselves, being recognized by receptors of immune cells, which consequently modulate host immunity beneficially [45, 101].

Immunomodulatory effect of prebiotics has been evaluated in vitro and in vivo. For instance, in an in vitro study, the influence of a FOS-inulin prebiotic at 200 μg/ mL on the ability of the chicken macrophage HD11 cell line to phagocytose and kill *Salmonella enteritidis* was tested. The influence on their ability to express selected inflammatory cytokines and chemokines, such as for IL-1β, lipopolysaccharideinduced TNF factor (LITAF), C-C motif chemokine ligand 4 (CCL4) and inducible nitric oxide synthase (iNOS), and nitric oxide production, was also evaluated. Results showed that phagocytosis of SE by macrophages was not affected with the FOS-inulin treatment, but there was a significant reduction of viable intracellular SE in macrophages treated with the prebiotic. On the other hand, prebiotic treatment did not influence the nitric oxide production, thus suggesting that the FOSinulin-mediated bacterial clearance was not mediated by this compound. Similarly, prebiotic treatment has no influence on expression of LITAF, CCL4, nor iNOS; however, IL-1β expression was significantly lower in macrophages treated with FOS-inulin, suggesting that this prebiotic can modulate the innate immune system by preventing IL-1β-associated macrophage cell death [102].

In a more recent study, GOS prebiotic was in ovo administered to evaluate the modulation of chicken intestinal microflora and demonstrate the molecular responses of the host animal. The study was performed on meat-type chickens, with 3.5 mg GOS delivered by in ovo injection on day 12 of egg incubation, and the analysis of microbial communities and mucosal gene expression was performed at day 42 post-hatching. Results showed that GOS increased the relative abundance of *Bifidobacterium* in the cecum. GOS also upregulated cytokine and barrier function genes in the jejunum and cecum, host defense peptides in the cecum, and free fatty acid receptors in the jejunum, ileum, and cecum, as shown in **Figure 8**, so that it has been demonstrated that GOS prebiotics have a bifidogenic effect in adult chickens, modulating gene expression related to intestinal immune responses and gut barrier function [32].
