Antibiotics

Antibiotics have been used in animals for therapeutic and growth-supporting prophylactic purposes since the 1940s. The use of antimicrobials reduces total bacterial load and pathogen pressure, modulates the immune system, and modulates the gastrointestinal microbiota, affecting the intestinal microbiota, immune response, and performance, and provides benefits to animal health. The direct effects of antibiotics on microbial populations or as growth factors are not clearly defined and may vary between products and applications. However, the age of the bird has a greater effect on intestinal maturation than drug use, and antimicrobials are more effective on less common species than on more common ones. Antibiotics cause significant species changes in the microbiota, but do not change the functionality of the microbiota. Beneficial effects of antibiotics include thinner epithelium formation, increased absorption of nutrients by the intestinal wall due to reduced microbial utilization, and increased nutrient delivery to host tissues due to more efficient nutrient utilization. It has been claimed that the irregular use and excessive use of antibiotics lead to the formation of bacterial resistance. Bacterial resistance poses a threat to human and animal antibiotic treatments due to the transmission of genes developed for antibiotic resistance to new generations of microorganisms. While their use as prophylactic doses in animal feeds has been banned in some regions, such as the European Union (EC Regulation No. 1831/2003), strict regulations on their use in animal husbandry or gradual bans are being considered in other regions. Antibiotics are also known for their energy loss reduction and anti-inflammatory roles. In recent years, many studies have been conducted on the most important effect of AGPs as nonantibiotic - anti-inflammatory.

Probiotics

Direct-fed microbial (DFM) probiotics are single or mixed cultures of live nonpathogenic microorganisms that provide host health benefits when administered in adequate amounts. The bacterial species currently used in probiotics are lactic acid bacteria (LAB – L. bulgaricus, L. acidophilus, L. casei, L. lactis, L. salivarius, L. plantarum), Streptococcus thermophilus, Bifidobacterium spp., Enterococcus faecium, and E. faecalis. In addition to bacteria, fungi such as Aspergillus oryzae and yeasts such as Saccharomyces cerevisiae are also used as probiotics. Their effects can be seen as competitive exclusion, promoting intestinal maturation and integrity, regulating the immune system, preventing inflammation, improving metabolism, supporting growth, improving fatty acid profile, and neutralizing enterotoxins. It has been reported that when the diet is supplemented with DFM together with multienzymes in broilers, the metabolizable energy and protein digestibility of high-fiber diets are increased. In addition, it has been proven that probiotics can improve the diversity of intestinal microbiota. Specifically, Bacillus spp. supports body weight gain; Pediococcus pentosaceus has a higher SCFA content. In addition, it has been shown that the density of Bacteroidetes in the cecal microbiota is directly related to the content of propionate, butyrate and isobutyrate, while the density of Firmicutes positively affects the production of acetate in the ceca. Regarding the immune response, it has been reported that L. acidophilus induces T-helper cytokines in caecal tonsil cells of chickens, while L. salivarius induces an anti-inflammatory response more effectively.

Prebiotics

Prebiotics are non-digestible feed ingredients responsible for selectively altering the composition and metabolism of intestinal microbiota. Prebiotics have the ability to increase the number of bifidobacteria and other species that positively affect the health of the host. It has been determined that poultry fed with β-glucan exhibit anti-Salmonella properties with immunomodulation that helps to increase immunity by increasing the number of IgA secreting cells, IgG levels and goblet cells during Salmonella challenge. Prebiotics also increase the number of LAB that help in the competitive exclusion of pathogens in the intestine (136). In addition, they help in the development of defense mechanisms. However, the mechanisms that help defense are not clear. Possible mechanisms are thought to be increasing the production of SCFAs that cause an acidic environment in the intestine, suppressing pathogens and also regaining some of the energy lost in competition with bacteria. Rapid clearance of pathogens due to prebiotic application is considered as a mechanism that increases immunity. The addition of slowly digestible prebiotics provides fermentable carbohydrates for the microbiota in the distal colon, which suppresses putrefaction. Dietary prebiotic supplementation benefits poultry performance and energy use. Commonly used prebiotics are inulin, fructooligosaccharide (FOS), mannanoligosaccharide (MOS), galactooligosaccharide, soy-oligosaccharide, xylo-oligosaccharide, pyrodextrin and lactulose. FOS is preferred for Bifidobacteria, which bind to the intestinal mucosa and prevent pathogenic bacteria, while MOS binds pathogens and ensures their excretion with the digestive content.

Phytosubstances

Phytosubstances are plant-based, natural bioactive compounds that can be added to feed to improve animal performance and health. Phytosubstances represent a wide range of bioactive compounds that can be extracted from various plant sources such as herbs and spices. The active compounds of phytosubstances are mostly secondary plant components such as terpenoids (mono- and sesquiterpenes, steroids, etc.), phenols (tannins), glycosides and alkaloids (alcohols, aldehydes, ketones, esters, ethers, lactones, etc.). Based on biological origin, formulation, chemical description, and purity, phytosubstances have been classified as: (1) herbs (products obtained from flowering, non-woody, and non-perennial plants), (2) botanicals (whole or processed parts of a plant, e.g., root, leaves, bark), (3) essential oils (distilled extracts of volatile plant compounds), and (4) oleoresins (essential oil-rich resins found in some plants). The possible mechanisms of action of phytosubstances on poultry health are as follows:

(1) Disruption of pathogen cell membranes by modulating the cell membrane of microorganisms,

(2) Change of virulence properties by affecting microbial cell surface properties by increasing the hydrophobicity of microbial species,

(3) Stimulation of growth of commensal bacteria such as lactobacilli and bifidobacteria in the intestine,

(4) Immune system stimulating effect

(5) Protection of intestinal tissue from microbial attack

(6) It is thought to have an anti-inflammatory effect.

Various oils, including carvacrol and thymol obtained from thyme plant and eugenol obtained from clove plant, have been shown to inhibit a wide range of pathogenic bacteria. Many controlled studies have shown the effects of oils used as feed additives on reducing various pathogens such as Salmonella, E. coli, Campylobacter and C. perfringens.

Organic acids

Organic acids are normal components of plant and animal tissues. Previously, organic acids were used as preservatives to extend the shelf life of foods because they control microbial contamination. These include lactate, acetate, propionate, butyrate, tannic acid, fumaric acid, and caprylic acid. These acids play a beneficial role in the intestinal health and performance of poultry. For example, they have been reported to increase the number of LAB in the ileum and ceca of broilers. They can also be produced in the host intestine after carbohydrate fermentation, especially in the ceca, where the microbial population and diversity are at their highest. Each of these acids is used in different ways in the host's body. Acetate is transported to the liver as an energy source for muscle tissue; propionate is converted to glucose in the liver by the process of gluconeogenesis; butyrate in the enterocytes of the small intestine serves as a vital energy source for host metabolic activities, helping with proliferation and development. However, butyrate does not always have positive effects; and this depends largely on its location and concentration in the GIT. Organic acids increase pepsin activity by lowering chyme pH. Peptides obtained from pepsin proteolysis aid growth because they can increase protein digestion by triggering the release of the hormones gastrin and cholecystokinin. They can cause increased body weight gain and feed conversion, reduced cumulative feed intake, suppression of bacterial cell enzymes, and reduction of pathogens such as Enterobacteriaceae spp. and Salmonella spp. The addition of organic acids can affect the cell membrane or cell macromolecules or can lead to bacterial death by inhibiting nutrient transport and energy metabolism. The antimicrobial activity of these compounds in the intestine depends on the ability of the acids to convert from undissociated to dissociated forms, their pKa value, and their hydrophobicity. If their supplements are not administered at appropriate doses, they can cause a decrease in villus height and width and crypt depth.

Bacteriophages

Bacteriophages (phages) are defined as specific intracellular parasites that reproduce using the metabolic machinery of bacteria. There are two major types of phages: virulent phages, which have a lytic life cycle; and temperate phages, which have a lysogenic life cycle. In a lytic life cycle, the phage recognizes specific bacteria, injects its genetic material, and then uses the host's metabolic machinery to replicate and assemble copies of itself. A process of cellular lysis mediated by the phage then releases the virions that are collected inside the cell. Once released, these new virions can infect another cell and begin the cycle again. In contrast, in a lysogenic life cycle, the phage recognizes the host bacterial cell, the injected DNA is incorporated into the bacterial genome, and it replicates with it. Under certain conditions, this DNA can detach itself from the genome and initiate a lytic life cycle. As it leaves the bacterial genome, the phage's DNA takes with it information that can be passed on to its next host. This process can cause the transfer of undesirable characteristics such as virulence factors or antibiotic resistance genes to the new host. Phage therapy is defined as the use of phages to treat bacterial infections and is limited to the use of virulent phages. As soon as these viruses were identified in 1915, their application in humans began. However, with the discovery of penicillin, its use became limited to some former Eastern Bloc countries. Today, the emergence of drug-resistant bacteria has led to a renewed focus on bacteriophages as a natural, non-toxic alternative treatment for bacterial infections. The advantages of phage therapy include the ability to target a specific bacterial group in treatment and, as a result, the normal microbiota is not affected, thus reducing the risk of secondary infection due to antibiotic treatments. Phages are thought to be more effective than antibiotics because they multiply only in the environment where their specific hosts are present, i.e., they have the ability to increase their density on site. Similarly, when the concentration of the host decreases following infection, the population of phages also decreases. Another important advantage is that phages can also be effective against antibiotic-resistant strains of sensitive bacteria. Phages can be used to control Salmonella and Campylobacter in broiler chickens.

Exogenous enzymes

Enzymes are special proteins that catalyze or accelerate chemical reactions. Enzyme activity is substrate-dependent or shaped by specific parts of the substrates, such as fat, protein or carbohydrate. Exogenous enzymes commonly used in poultry diets are β-glucanase, xylanase, amylase, α-galactosidase, protease, lipase and phytase. The role of exogenous enzymes is to replace the functions of endogenous enzymes to counteract anti-nutritional factors found in conventional and unconventional poultry diets. These exogenous enzymes are used in combination with unconventional ingredients to reduce feeding costs and efficiently use unconventional feed ingredients. Because unconventional feed ingredients are typically rich in fiber, endogenous enzymes are insufficiently utilized and cannot be used by the poultry. Accordingly, non-starch polysaccharide (NSP) degrading enzymes that produce oligosaccharides can also reduce putrefaction in the cecum, since bacteria will prefer carbohydrates for fermentation when both carbohydrates and proteins are present in the intestine. Carbohydrate supplementation increases the ratio of lactic and organic acids, reduces ammonia production, and increases the concentration of NSP hydrolysis indicator, which supports the growth of beneficial bacteria. Multi-enzyme supplementation, such as xylanase, amylase, and protease, optimizes fiber utilization and enables broilers to achieve better growth performance. Another important effect of enzyme use is the prebiotic effect it creates on the intestinal microbiota. This mechanism is explained by the use of arabinoxylan oligosaccharide (AXOS) and xylooligosaccharide (XOS) components, which emerge as a result of the degradation of the arabinoxylan structure as a result of xylanase activity, by Firmicutes bacteria in the last part of the intestine as prebiotic effective components. As is known, the last part of the intestine is a risky area where pathogenic bacteria are densely colonized. Although there are various types of beneficial microorganisms in the poultry microbiota, the most important are the Firmicutes group bacteria. Studies show that as the number of Firmicutes group bacteria increases, butyric acid synthesis from prebiotic compounds occurs, and colonization of pathogenic bacteria in the region becomes quite difficult.

Special Topic: The Link Between Gut Microbiota and Performance

Factors affecting microbiota composition can alter productivity and explain, at least in part, the performance variability observed within individual flocks. Some studies have directly linked changes in microbiota to chicken performance. Poultry productivity can be measured by different parameters, including feed conversion ratio (FCR), residual feed intake (RFI), body weight gain (BWG), metabolizable energy (AME), and time to slaughter weight.

The seasonal changes in the broiler microbiota throughout the life cycle were described by Rinttilä and Apajalahti. A recent study using mass sequencing determined the microbiota composition at days 7, 14, and 42. In short, on day 7, the microbiota was dominated by 3 species of the order Clostridiales (Flavonifractor, Pseudoflavonifractor, and Lachnospiracea). These 3 species are considered responsible for converting polysaccharides into SCFAs. On day 21, the Faecalibacterium genus, which has anti-inflammatory properties, is dominant. On day 42, Faecalibacterium continues to dominate despite the increase in the proportion of the Roseburia genus. This genus is defined as a saccharolytic bacterium that produces butyrate. In addition, on day 42, the density of other SCFA-producing bacteria such as members of the Lachnospiraceae incertae sedis family and the Oscillibacter genus increases.

Although they are closely related, each section of the chicken gastrointestinal tract has a different microorganism profile. The results of microbiota studies are affected by the unique characteristics of the birds such as genetics, gender, age, breed, health status, as well as farm conditions such as diet type, feed additives, environment, and farm management.

By pyrosequencing the V3 region of the 16S rRNA gene, it was determined that bacterial communities that produce butyrate and degrade cellulose and starch in the cecum were associated with high-performance chickens (CE=1.32). Clostridium islandicum, Ruminococcus sp., Bacteroides fragilis and Lactobacillus coleohominis are included in this beneficial bacterial group. In a study where the V3 region of the 16S rRNA gene was amplified and DNA sequenced using an Illumina MiSeq sequencer, 4 OTUs were identified that were more intense in high-performance chickens. In another DNA sequencing study, the V4 region of the 16S rRNA gene was amplified using an Illumina MiSeq sequencer in order to correlate some bacterial groups with chicken body weight. In the cecum, Akkermansia, Prevotella and Anaerovibrio negatively affected body weight gain, while Lactococcus showed a positive correlation.