Literature Review
Basics overview of essential oils and their antibacterial mechanism
EOs are liquid aromatic fluid compounds from plants such as the leaves, stems, roots, flowers, seeds or fruits by steam distillation solvent extraction, steam distillation or extrusion
Essential oils are primarily composed of complex compounds, including terpenes and their oxygenated derivatives known as terpenoids, as well as aromatic and aliphatic acid esters and phenolic compounds According to Hammer and Carson, terpenoids are the most prevalent constituents of essential oils, while phenyl-propanoids, although less common, also play a significant role.
Essential oils consist of 85% main ingredients, with minor components contributing 2 to 3 key ingredients that define their essential characteristics For instance, thymol and carvacrol, which make up approximately 80% of oregano's essential oils, are significant phenols known for their antibacterial and antioxidant properties Additionally, oregano contains other compounds like r-cymene, although this particular ingredient is not as prominent.
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18 effective against bacteria, it creates favourable conditions for carvacrol transport through the cytoplasmic membrane [22]
Essential oils (EOs) possess unique chemical structures and biological effects that vary based on factors such as species, climate, ecology, harvesting time, and isolation methods Approximately 3,000 plant families, including Alliaceae, Apiaceae, Asteraceae, Lamiaceae, Myrtaceae, Poaceae, and Rutaceae, have been identified for their medicinal properties Notable plants known for their essential oils include black pepper, garlic, cinnamon, anise, thyme, turmeric, and oregano.
Supplementing poultry and pigs with essential oils has been shown to modulate microbiota and enhance performance, leading to greater average daily gain (ADG) Research indicates that essential oils improve feed digestion by boosting bile salt secretion and stimulating enzymatic activities in the pancreatic and intestinal mucosa The antimicrobial properties of essential oils are linked to their chemical structure and diverse mechanisms of action.
Essential oils, due to their hydrophobic nature, significantly alter cell membranes by penetrating and disrupting the phospholipid bilayer, which increases membrane permeability This disruption causes leakage of ions and protons, ultimately affecting the internal environment of bacterial cells Microscopic studies indicate that even low concentrations of certain essential oils can create holes in the cell walls of sensitive bacteria, such as C perfringens, particularly in their vegetative and lysed forms.
Carvacrol disrupts the osmotic balance and alters the intracellular pH of bacteria by facilitating ion transport across the membrane The undissociated form of carvacrol penetrates the cytoplasmic membrane, acting as an H+ proton within the bacterial cytoplasm, where it combines with a K+ ion and exits the cell, carrying positive ions with it This process acidifies the cytoplasmic environment, negatively impacting the membrane's proton pump mechanism and ultimately affecting ATP production within the cell.
(3) Changes in cellular metabolism: For example, thymol has been shown to disrupt citrate metabolism in Salmonella (reviewed by F Nazzaro) [36]
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Cinnamaldehyde, a compound found in cinnamon, has been shown to inhibit the communication systems between bacteria by disrupting the synthesis of quorum sensing molecules This action limits the pathogenic properties of microorganisms, reducing the growth and aggregation of both Gram-positive and Gram-negative bacteria Consequently, it decreases biofilm formation and the expression of various virulence factors, as well as protein hydrolysis activity, thereby enhancing antibacterial effectiveness.
(5) Other mechanisms of essential oil might be linked to nutrient absorption inhibition, enzymatic inhibition, DNA, RNA, and protein synthesis by bacterial cells [31][38]
Figure 1 - 1 The essential oils' mechanism of action and target locations on microbial cells
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Basic overview of acidifiers and their antibacterial mechanisms
In addition to essential oils, various acids and their salts are increasingly used as alternatives to antibiotics in animal feed, with organic and fatty acids exhibiting both antibacterial properties and growth-promoting effects in livestock and poultry These organic acids can be categorized based on the length of their carbon chains—short, medium, or long—or by their saturation level, which can be either saturated or unsaturated Notably, some organic acids serve as intermediaries in the tricarboxylic acid (TCA) cycle, playing a crucial role in generating energy for various biological functions.
Some antimicrobial activity has been shown for different acidifiers and organic acids
Organic acids (OAs) added to animal and poultry feeds significantly reduce Salmonella levels due to their strong antibacterial properties For instance, benzoic acid not only exhibits antibacterial effects but also improves weanling piglet efficiency, resulting in a 12% increase in average daily gain (ADG) The antibacterial action of OAs involves lowering pH and their ability to penetrate bacterial cells, leading to a reduction in intracellular pH that disrupts microbial metabolism by inhibiting essential enzymes This process can cause bacterial cells to deplete energy reserves, ultimately leading to cell starvation Additionally, OAs lower feed buffering capacity, which increases hydrochloric acid production in the stomach and enhances nutrient digestibility A decreased pH also boosts pepsin activity and stimulates pancreatic enzyme secretion, further improving nutrient absorption.
Organic acids (OAs) such as butyric acid, benzoic acid, citric acid, propionic acid, fumaric acid, formic acid, phenyllactic acid, and lactic acid are commonly used in poultry feeds Research by Fascina et al indicates that acetic acid, lactic acid, citric acid, benzoic acid, and formic acid can significantly improve feed efficiency and growth in broiler chickens Additionally, the inclusion of coated sodium butyrate has been shown to enhance growth efficiency in broiler chickens, primarily due to increased mucosal production Therefore, supplementing poultry diets with organic acids can lead to improved growth performance.
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21 efficiency of the birds by improving the uptake of available nutrients and decreasing the harmful metabolites from gut microbiota [50]
Organic acids (OAs) serve as effective nutritional food preservatives by inhibiting microbial growth and extending shelf life They can function as acidifiers, antioxidants, antifungals, flavor enhancers, and pH modifiers in both animal feed and human food applications However, some microorganisms are developing resistance to certain OAs, similar to antibiotic resistance, highlighting the need for new OAs with antimicrobial and immunomodulatory properties suitable for broiler diets and safe for human consumption Research indicates that adding phenyllactic acid (PLA) to broiler chicken diets enhances growth performance and feed conversion ratio (FCR), while also improving immunological properties and increasing lactic acid-producing bacteria Moreover, the inclusion of PLA reduces coliform bacteria and enhances chicken meat quality, suggesting that dietary PLA could be a viable alternative to antibiotics in poultry nutrition.
Hexanoic acid, or caproic acid, is utilized in poultry and pig feeds, demonstrating efficacy in reducing Salmonella spp in chickens, similar to medium-chain fatty acids (MCFAs) Research by Fascina et al indicated that broilers receiving a blend of organic acids—benzoic, lactic, citric, formic, and acetic acids—showed enhanced performance compared to those without supplementation Additionally, this supplementation led to increased expression of crucial proteins such as ZO-1, LEAP-2, claudin-1, claudin-4, occludin, and mucin.
2 were observed in the jejunum when sodium n-butyrate was used [16]
The primary mechanism of organic acids:
Organic acids penetrate bacterial cells in their undissociated form, but as the internal pH rises, acid dissociation occurs, releasing H+ ions and lowering the pH This triggers the H+-ATPase pump, which consumes energy to restore normal pH levels, ultimately reducing energy availability for cell proliferation and disrupting DNA replication and various metabolic processes Consequently, bacterial cells expend energy to counteract the pH drop, leading to exhaustion and cell death Additionally, the presence of anions (-COOH) negatively impacts critical metabolic functions, impairing glycolysis, hindering active transport, and obstructing signal transduction due to the low internal pH.
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Figure 1 - 2 Organic acid action mechanisms in bacterial cells (internet)
The effect of a combination of essential oils and acidifiers and their mechanism
Bassole and Juliani [55] found that a combination of thymol, carvacrol, and eugenol at lower concentrations exhibited additive or synergistic antimicrobial properties Conversely, other research indicated that the inclusion of essential oils (EOs) or organic acids (OAs) in broiler feed did not significantly impact growth efficiency or intestinal absorption [56,57] This inconsistency may be attributed to the instability and variability of these compounds during storage, feed processing, and digestion Consequently, some studies have suggested that merging essential oils with organic acids could enhance their beneficial effects.
Organic acids enhance the efficacy of essential oils by transforming their active components from a dissociated to a molecular form, which can easily penetrate bacterial cell walls and cause damage Conversely, essential oils can disrupt bacterial cell walls, allowing organic acids to enter and interfere with bacterial function This interaction is particularly effective in slightly acidic to neutral pH environments, such as the small and large intestines, where organic acids are often dissociated and unable to penetrate bacterial cell walls Research has shown that blends of essential oils and benzoic acid can improve average daily gain (ADG) and feed conversion ratio (FCR) in broiler chicks, highlighting the synergistic effects of these compounds in promoting poultry health.
Essential oils (EOs) have a lipophilic structure, allowing them to penetrate bacterial cell walls, particularly when benzoic acid is undissociated This penetration disrupts the bacteria's cellular homeostasis as they attempt to counterbalance the influx of H+ ions resulting from the Na+/K+ pumping mechanism, leading to a bactericidal effect.
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The bacteriostatic effect is defined by a mechanism that actively depletes bacteria, diminishes their pathogenicity, and disrupts vital processes The synergistic combination of essential oils and benzoic acid can effectively damage the cell walls of pathogenic organisms by easily permeating and dissociating them.
Impact of EOs, OAs and EOAs on growth performance
3.1.1 Impact of EOs on growth performance
Essential oils (EOs) are recognized as growth promoters in poultry, although results from animal trials can vary significantly due to factors like the type of essential oils, animal species, diets, and environmental conditions Research indicates that a combination of thymol and carvacrol can enhance growth performance in broiler chickens, leading to improved body weight gain and feed efficiency Additionally, a mixture of 25% thymol and 25% carvacrol has shown positive effects on birds challenged with C perfringens Other studies have demonstrated that EOs containing carvacrol, cinnamaldehyde, and capsicum can boost production performance and feed consumption in broilers Furthermore, incorporating thymol and cinnamaldehyde into diets has been linked to increased body weight gain in broiler chickens The use of thyme as an EO has also correlated with improved growth performance in C perfringens-challenged birds A dietary blend of essential oils, including carvacrol, cinnamaldehyde, and capsicum oleoresin, has been shown to enhance growth and feed efficiency In pigs, dietary supplementation with carvacrol and thymol resulted in significant body weight gain, and a combination of carvacrol, capsicum oleoresin, and cinnamaldehyde improved final body weight, weight gain, and feed conversion ratio compared to untreated controls.
Some studies have shown that thymol and carvacrol products do not impact growth performance in broiler chickens infected with necrotic enteritis (NE) Furthermore, there were no significant differences in feed consumption, weight gain, or feed-to-gain ratio between chickens that received a blend of thymol and cinnamaldehyde and those that did not.
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24 oregano oil, carvacrol, thyme oil, eucalyptus oil, and thymol did not increase growth efficiency [71]
3.1.2 Impact of OAs on growth performance
Organic acids have long been used to preserve feed and possess antimicrobial properties that can aid in disease control and enhance growth performance Research indicates that feeding organic acids (OAs) such as lactic, benzoic, formic, citric, and acetic acid—comprising 30.0%, 25.5%, 7%, 8%, and 6.5% respectively—can significantly improve poultry growth efficiency and serve as an effective antibiotic alternative Additionally, supplementing chicken diets with sodium butyrate has been shown to stimulate growth performance, improve feed conversion ratios, and increase body weight and average daily gain.
Incorporating 0.2 % protected organic acids into the diet of pigs can boost growth rates Protected organic acid rations fed to piglets increased average body gain over a period of 0-2 weeks and throughout the entire experimental period (0–6 weeks) [73] The results were identical to those of the authors' [74] , but for the lack of impact on broiler feed consumption in the former study S I LEE [75] showed that OAs diet with citric acid (13%), fumaric acid (17%), malic acid (10%), MCFA (1.2%), as well as capric and caprylic acid significantly altered the production of eggs, egg stability while increasing the fecal Lactobacillus and reducing E coli in the gut On the other hand, the other researcher found that OAs did not enhance growth or intestinal morphology [48]
3.1.3 Impact of EOAs on growth performance
Previous studies indicate that the combination of essential oils (EOs) and organic acids (OAs) enhances growth performance in broiler chickens Gheisar M reported that a mixture of thymol, vanillin, citric acid, and sorbic acid improved growth rates significantly Additionally, Gao found that dietary supplementation with EOs and OAs led to increased average daily gain (ADG) and improved gain-to-feed ratio (G/F) compared to control diets Liu's research revealed that incorporating EOs and OAs into broiler diets reduced feed intake from day 22 to 42, while also decreasing feed-to-gain ratio (F/G) throughout the entire 42-day study.
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25 acid increased body weight by 2 and 1.4% in broilers at 21 d and 42 d, respectively [61] BW gain was significantly improved in a different study when sodium butyrate was combined with ginger oil and carvacrol [78]
The combination of essential oils (EOs) and organic acids (OAs) significantly enhanced average daily gain (ADG) and feed conversion ratio (FCR) in broiler chickens by 42 days of age Additionally, birds receiving the OAs and EOs mixture exhibited superior body weight gain (BWG) compared to those treated with antibiotic growth promoters (AGPs) and infected groups during the growth period A study conducted in Spain revealed that a diet incorporating short-chain fatty acids (SCFA), medium-chain fatty acids (MCFA), and specific organic acids, along with essential oils like thymol, carvacrol, and cinnamaldehyde, led to improved growth performance in broiler chickens challenged with C perfringens at 42 days.
Beside, in weaned pigs, a mixture of thymol, carvacrol, benzoic acid, calcium formate and fumaric acid (25%, 25%, 50%, 3%, 1%, respectively) improved performance
A Brazilian study utilizing a pig model infected with E coli found that a combination of benzoic acid, thymol, 2-methoxyphenol, eugenol, piperine, and curcumin significantly enhanced average daily gain (ADG) and body weight (BW) throughout the experimental period Additionally, from days 21 to 35, the study reported improved ADG and reduced feed-to-gain ratio (F/G) when comparing the negative control group to the colistin-treated group.
Some studies indicate that the inclusion of essential oils (EOs) and organic acids (OAs) in broiler diets does not significantly impact growth efficiency Specifically, research has shown that broilers' growth performance remained unchanged with EOs and OAs supplementation at concentrations of 30 and 2,000 ppm.
Impact of EOs, OAs and EOAs on intestinal morphology
3.2.1 Impact of EOs on intestinal morphology
A healthy gut is crucial for optimal feed utilization and growth performance in livestock Research indicates that the supplementation of essential oils (EOs) can enhance intestinal health by improving villus height (VH) and VH/CD ratios For instance, adding 300 ppm of oregano essential oil to broiler diets significantly increased jejunal and ileal villi height Additionally, cinnamon bark oil (CNO) has been shown to positively affect villus height in the duodenum, jejunum, and ileum compared to control groups However, some studies reported no significant impact on villus morphometry.
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26 observed in an experiment that used a combination of essential oils (including carvacrol, cinnamaldehyde, and capsicum oleoresin) [90]
3.2.2 Impact of OAs on intestinal morphology
Research by Panda et al [91] demonstrated that butyrate, regardless of dietary concentration (0.2%, 0.4%, or 0.6%), significantly increased the villus height (VH) and crypt depth (CD) in the duodenum Additionally, the inclusion of organic acids, such as butyric, lactic, and fumaric acid in equal ratios of 3% each, further elevated VH in the duodenum and jejunum of broiler chickens [92] Moreover, a combination of sorbic and citric acid at concentrations of 1.0% and 0.2%, respectively, was found to enhance both the villus width and height in the small intestine of broiler chickens at 14 days of age [93].
Sodium butyrate has been shown to enhance villus height in broilers nine days after infection with C perfringens, as reported by Song [16] Additionally, a combination of propionic and formic acid has been found to improve ileal morphology [94] Another study indicated that organic acids (OAs) significantly increased ileal villus height on day 42 compared to the negative control group [72].
3.2.3 Impact of EOAs on intestinal morphology
Research indicates that the combination of sodium butyrate with ginger oil and carvacrol significantly enhances villus height and the VH/CD ratio Additionally, chicks fed diets containing a mix of formic and propionic acids, carvacrol, cuminaldehyde, and eugenol demonstrated increased ileal villus height and reduced crypt depth by 42 days of age compared to those on a control diet Similarly, a blend of sorbic acid, fumaric acid, and thymol improved crypt depth in both the jejunum and ileum from 22 to 42 days of age Furthermore, a group receiving caproic acid and cinnamylaldehyde exhibited a superior VH/CD ratio in the ileum compared to the positive control group.
Research indicates that the inclusion of essential oil additives (EOAs) such as thymol, eugenol, and curcumin at a dosage of 4 g/kg enhances villus height in the chicken cecum A study by N Abdelli found that EOAs supplementation significantly improved gut histomorphology in broilers 42 days post-infection with C perfringens Additionally, a trial demonstrated an increased villus height to crypt depth (VH/CD) ratio at 21 days of age when a blend of EOAs, including citric acid and vanillin, was used Furthermore, laying hens receiving EOAs also exhibited heightened ileum villus height by week 30.
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27 in the duodenum and jejunum was also increased by feeding a combination of OAs and EOs (benzoic acid, calcium formate, fumaric acid, thymol and carvacrol) in weaned pigs [82]
Several studies indicate that there is no significant difference in ileal crypt depth and villi height in broiler chickens when comparing essential oils (EOs) and organic acids (OAs) Additionally, the inclusion of OAs and phytogenic additives did not show a noticeable impact on villus height or crypt depth in these chickens.
Impact of EOs, OAs and EOAs on antibacterial activity and intestinal lesions
3.3.1 Impact of EOs on antibacterial activity and intestinal lesions
Recent studies have shown that the application of essential oils leads to positive regulation of the microbial population and supports digestive functions According to Mitsch
A blend of essential oils (EOs) containing thymol and carvacrol significantly reduced the presence of C perfringens in the jejunum and cecum of chickens Additionally, the EOs group showed a notable decrease in ileal and cecal E coli counts compared to the control group, while Lactobacilli levels remained unaffected Furthermore, another EOs blend influenced the caecal microbiota, leading to increased concentrations of E coli and Lactobacillus by 41 days of age in the birds.
C.perfringens, macroscopic gut lesions were not observed in those fed with EOs mixture
A diet containing 25% thymol and 25% carvacrol, administered at dosages of 60, 120, or 240 mg/kg, significantly reduced NE-related small intestine lesion scores and decreased E coli concentrations in the ileum Additionally, this essential oil combination effectively alleviated the severity of intestinal lesions caused by C perfringens infection Broiler chickens receiving a mixture of thymol and carvacrol at a dosage of 200 mg/kg exhibited lower levels of Escherichia coli and Clostridium perfringens, while Lactobacilli populations increased compared to the control group.
At 35 days of age, a low dose of encapsulated carvacrol decreased C perfringens in the ileum of birds and alleviated NE lesions in the intestines [99] Using the EOs (thyme (Thymol as ≥ 40%) and clove EOs (Eugenol as ≥ 55%) decreased C.perfringens populations in the intestines and significantly decreased gross gut pathology in broiler chickens when infected with C perfringens [66] Besides, CNO (cinnamon bark oil) increased the number of cecal E.coli but did not influence Lactobacillus counts compared to the control group [89] Previous research has shown that carvacrol and thymol can decline the number of E.coli and
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C.perfringens in the intestines of broilers while increasing the amount of Lactobacilli [100,101] After using EOs mixture (thymol and carvacrol 25%:25%, respectively) were added, the number of Lactobacillus crispatus and Lactobacillus agilis significantly increased in the ileum, while Lactobacillus salivarius and Lactobacillus johnsonii counts were significantly reduced [102] Moreover, intestinal lesions were alleviated, and the virulence factors of pathogenic bacteria were reduced
3.3.2 Impact of OAs on antibacterial activity and intestinal lesions
Organic acids negatively influenced pathogenic bacterial loads, including coliforms and Clostridia in the ileum compared to AGPs treatment [103] E.coli, Lactobacillus, and
C.perfringens cecal populations were decreased when an organic acid blend was added to the diet [46] The number of Salmonella enteritidis in the cecum and liver were reduced by supplementing with caprylic acid (CA) at 0.7 or 1% [104] In the hens, dietary supplementation with OAs mixture of microencapsulated containing MCFAs increased Lactobacillus fecal content while lowering E.coli in the fecal content [75] Similarly, Lactobacillus counts increased with OAs and MCFAs addition, while the number of E.coli reduced in ileal [105] The same, cecal lactobacilli were increased and cecal E coli was reduced when a mixture of formic and propionic acid was used [94]
Sodium butyrate supplementation effectively reduced E coli and C perfringens counts in the cecum, as well as the liver levels of C perfringens compared to the positive control group Additionally, in weaned piglets, the counts of Lactobacilli in feces were significantly higher in groups receiving organic acids (OAs), essential oils (EOs), and a combination of OAs and EOs than in the positive control groups.
3.3.3 Impact of EOAs on antibacterial activity and intestinal lesions
Researchers suggest that the combination of essential oils and organic acids enhances livestock performance more effectively than using these ingredients separately They assert that essential oils improve the absorption of organic acids, resulting in a significant decrease in harmful bacteria proliferation.
Numerous studies indicate that essential oils (EOs) and organic acids (OAs) possess significant antimicrobial properties Notably, the combination of acetic acid and thymol (or carvacrol), as well as citric acid with thymol (or carvacrol), effectively reduced populations of Salmonella typhimurium Additionally, mixtures of EOs, such as carvacrol and ginger oil, or sodium butyrate, further enhance these antimicrobial effects.
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A study found that 29 essential oils significantly reduced gross pathological and histopathological lesion scores compared to a control group Additionally, a mixture of essential oils, including thymol (1.7%) and vanillin (1.0%), along with organic acids like citric acid (25%) and sorbic acid (16.7%), positively impacted beneficial bacteria such as Lactobacilli Research by H Basmaciolu-Malayolu indicated that birds fed diets containing essential oils had lower populations of E coli in the ileum compared to those on a control diet Furthermore, the combination of organic acids and essential oils resulted in a higher number of fecal Lactobacilli in weaned pigs compared to the control group.
Research indicates that the use of essential oil blends (EOAs) significantly reduces E coli and Salmonella levels in the intestines and cecal contents of birds A study by MA Gole demonstrated a decrease in E coli in birds compared to a control group Additionally, a mixture of essential oils and organic acids led to reduced numbers of these bacteria in broiler chickens at 21 and 70 days of age Furthermore, EOAs containing sorbic acid, fumaric acid, and thymol were effective in lowering E coli levels in broiler chickens Blends of organic acids and natural aromatic compounds also contributed to a reduction in fecal Enterobacteriaceae and C perfringens.
The addition of essential oils and benzoic acid did not significantly affect the levels of Lactobacillus spp and E coli in the ceca of broilers at 17 and 35 days of age Similarly, a study by L.M Rodrigues found that combining these compounds, including thymol, eugenol, piperine, 2-methoxyphenol, and curcumin, had no impact on microbial counts such as Bifidobacterium.
Lactobacillus spp., E coli, and coliforms totals) The broilers in the OAs+EOs group
Organic acids such as fumaric, sorbic, malic, and citric acids, along with essential oils like thymol, vanillin, and eugenol, exhibited the lowest macroscopic scores among all tested groups Additionally, a combination of thymol, piperine, eugenol, and benzoic acid significantly reduced lesion scores caused by various factors.
Impact of EOs, OAs and EOAs on antioxidant enzyme activity
3.4.1 Impact of EOs on antioxidant enzyme activity
Research indicates that a blend of essential oils (EOs), including ginger powder and thyme oil, significantly lowers malondialdehyde (MDA) levels in the liver, duodenal mucosa, and kidneys of broiler chickens Notably, at days 24 and 42, these EOs enhance the activities of superoxide dismutase and glutathione peroxidase while simultaneously reducing MDA concentrations in both serum and liver Additionally, a mixture comprising 5% carvacrol, 3% cinnamaldehyde, and 2% capsicum oleoresin has shown promising results in improving overall health parameters.
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Dietary essential oils (EOs) such as carvacrol, cinnamaldehyde, and capsicum oleoresin have been shown to enhance hepatic antioxidant concentrations, including carotenoids, total vitamin E, and coenzyme Q10 in broilers Additionally, oregano essential oil (OEO) has been found to reduce serum levels of malondialdehyde and nitric oxide while increasing superoxide dismutase concentrations and total antioxidant status in Eimeria-infected broilers Furthermore, research by S Chowdhury et al indicates that cinnamon bark oil supplementation can improve superoxide dismutase activity and cholesterol levels in the blood of chickens.
3.4.2 Impact of OAs on antioxidant enzyme activity
A study by A.M Abudabos revealed that a blend of organic acids significantly enhanced the total antioxidant capacity (TAC) in the serum of broiler chickens infected with Salmonella Additionally, the inclusion of benzoic acid in pig diets led to increased concentrations of SOD and GSH-PX in the jejunum mucosa However, organic acid supplementation did not affect MDA levels in the jejunum mucosa Furthermore, W.H Zhang reported findings related to varying doses of organic acids.
Supplementation of sodium butyrate at doses of 100 g/kg significantly altered the antioxidant status in broiler chickens infected with LPS Notably, SOD levels and catalase activities increased at 21 and 42 days of age, while MDA concentrations decreased at both time points Additionally, the activity of antioxidant enzymes such as CAT, T-AOC, and GSH-Px improved in the jejunal mucosa, accompanied by a reduction in NO content and iNOS activation in serum These findings align with those of W Wu et al., who reported that a sodium butyrate dose of 800 mg/kg enhanced T-AOC levels and lowered MDA proportions in the jejunal mucosa.
3.4.3 Impact of EOAs on antioxidant enzyme activity
Incorporating thymol, benzoic acid, and BMEO—a mixture of benzoic acids, thymol, eugenol, and piperine—into turkey diets significantly improved antioxidant status Specifically, BMEO elevated the levels of GSH-PX and GST while reducing MDA concentrations in the liver, thigh, and breast compared to the control group.
Impact of EOs, OAs and EOAs on immune responses
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The immune system plays a vital role in the survival of animals Research indicates that essential oils and organic acids can effectively stimulate the natural immune response in livestock and poultry.
3.5.1 Impact of EOs on immune responses
Thymol and carvacrol supplementation in broiler chickens significantly improved hypersensitivity responses, increased IgG anti-sheep blood cell titers, and reduced the heterophil to lymphocyte ratio compared to the control group Additionally, a blend of carvacrol, capsicum oleoresin, and cinnamaldehyde positively influenced immunomodulation and gut integrity Furthermore, incorporating essential oil blends, such as thymol and vanillin, along with organic acids like citric and sorbic acid, into broiler feed at day 70 led to enhanced s-IgA content.
Thymol enhances barrier function in epithelial cells by reducing reactive oxygen species (ROS) production and lowering pro-inflammatory cytokine mRNA levels Specifically, thymol downregulates cell permeability and decreases TNF-α and IL-8 mRNA concentrations while simultaneously upregulating zonula occludens-1 (ZO-1) and transepithelial electrical resistance (TEER) in response to Lipopolysaccharide (LPS) challenges.
3.5.2 Impact of OAs on immune responses
Emami et al highlight the beneficial effects of organic acids (OAs) on both humoral and cellular immunity Their research indicates that OAs supplementation can enhance antibody levels, including IgG against SRBC and total primary antibodies, as well as IgG, IgM against SRBC, and total secondary antibodies in broiler chickens challenged with ETEC Additionally, the combination of probiotics and organic acids alters TLR-2 and cytokine profiles, leading to a reduction in TLR expression in cecal tonsils.
2 and ileal IFN-γ, IL-12p35 mRNA expression at 11 day of ages while improving cecal tonsil IFN-γ and IL-6, IL-10 mRNA in ileal at 22 day of ages [93]
The combination of sorbic acid, fumaric acid, and thymol at a dosage of 0.30 g/kg resulted in an increased spleen weight in chickens and elevated IgA concentrations in the duodenal and ileal mucosa by day 42 Additionally, a blend of essential oils (EOs) with fumaric, citric, and malic acid at doses of 0.1% and 0.2% showed significant effects, with concentrations of 17%, 13%, and 10% respectively, alongside a 1.2% inclusion.
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MCFA (including: capric and caprylic acids)) enhanced the level of IgG, and lymphocyte % in pigs [117]
3.5.3 Impact of EOAs on immune responses
The EOAs (including thymol, fumaric acid and sorbic acid - minimum of 100, 200,
Supplementation with a mixture of essential oils (EOs) and organic acids (OAs) resulted in significantly higher secretory IgA concentrations in the duodenal and ileal mucosa compared to other groups This blend, which includes thymol, eugenol, and various acids, positively influenced claudin-1 mRNA expression in the jejunum By day 70, the specific combination of EOs and OAs, featuring thymol and vanillin, notably increased s-IgA levels in the jejunal mucosa Furthermore, broilers receiving this EOs and OAs combination demonstrated enhanced expression of mucin-2, occludin, and claudin-1 genes compared to the challenged control group Additionally, groups treated with TBA and CCA exhibited lower tumor necrosis factor α (TNF-α) levels than the positive control group, indicating a potential anti-inflammatory effect.
Supplementation with AB (benzoic acid and Bacillus coagulans), AO (benzoic acid and oregano oil), and AOB (a combination of Bacillus coagulans, benzoic acid, and oregano oil) effectively reduced serum levels of TNF-α and LPS in piglets challenged with ETEC ETEC infection led to increased levels of TNF-α and IL-1β in the mucosal jejunum and decreased sIgA levels; however, the addition of AOB countered these effects Additionally, compared to the ETEC group, AOB exhibited higher mRNA levels of mucin-2 and claudin-1, while showing reduced expression of NOD2 and TLR4 in the jejunal mucosa.
Impact of EOs, OAs and EOAs on intestinal microbiota
3.6.1 Impact of EOs on intestinal microbiota
Recent in vivo studies indicate that bioactive phenolic extracts (BPE) enhance the relative abundance of Firmicutes in the cecum Additionally, birds fed tannin showed increased levels of Clostridiales, Ruminococcaceae, and Lachnospiraceae while experiencing a reduction in Bacteroides at the genus level Furthermore, the inclusion of 25% thymol and 25% carvacrol at a dosage of 120 mg/kg also contributed to these microbial changes.
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Dietary supplementation with BEOs led to a decrease in the relative abundance of Firmicutes, Cyanobacteria, and Proteobacteria At the genus level, this supplementation reduced the proportions of Lactobacillus salivarius and Lactobacillus johnsonii while increasing the relative abundance of Lactobacillus crispatus, Lactobacillus agilis, and E coli compared to the control group.
N Zhu [121] demonstrated that the supplementation plant extracts (PEs) (including 20% carvacrol and 25% thymol using dose of 400 mg/kg) significantly decreased the number of Lactobacillus concentrations compared to control group Contrastly, the relative abundance of the Faecalibacterium and unclassified_Rikenellaceae in the high essential oil group increased According to the findings PEO (78.3% Cinnamicdehyde, 4% Isophorone, and eugenol (2.7%)) treatment enhanced the relative abundance of Bacteroidetes and reduced the abundance of Firmicutes at the phylum, as well as the level of Lactobacillus at the genus in the cecal microbiota of chickens The PEO group had a higher relative abundance of Alistipes, unclassified_Rikenellaceae, Roseburia, and Anaeroplasma than the control group [122] Furthermore, EOs could be to alter the intestinal microbial composition in weaned piglets [68] In fact, the EOs group had higher relative abundances of
Lactobacillales, Bacilli, Veillonellaceae and Streptococcaceae which is considered beneficial bacterial species
3.6.2 Impact of OAs on intestinal microbiota
Research indicates that the cecal microbiota of chickens contains a significant number of fiber-degrading bacteria and short-chain fatty acid (SCFA) producers The addition of sodium butyrate notably reduced these populations Furthermore, in chickens infected with Eimeria tenella, the relative abundance of Firmicutes was significantly lower in those receiving sodium butyrate compared to the infected group without it, while the abundance of Bacteroidetes was considerably higher in the ceca of these birds.
At the phylum, sodium butyrate increased the number of Firmicutes, Proteobacteria (400mg/kg) and Bacteroidetes (800mg/kg) At the family, sodium butyrate declined in
Supplementation of organic acids (OAs) significantly influences the microbial community composition in the cecal lumen, notably increasing the concentrations of Lachnospiraceae and Rikenellaceae while reducing Enterobacteriaceae levels Additionally, a dose of 800 mg/kg of Ruminococcaceae and a decrease in Lactobacillaceae to 400 mg/kg were observed Furthermore, OAs positively affected the populations of Bacteroidetes and Firmicutes at the phylum level, while diminishing the abundance of unclassified Coriobacteriaceae and unclassified Burkholderiales.
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34 increased the Caldanaerocella and Dorrea abundance at genus level when challenged with
The addition of organic acids, specifically formic acid (≥11%), acetic acid (≥5.1%), and propionic acid (≥10%), significantly altered the gut microbiota composition compared to the control group This intervention led to an increased proportion of Firmicutes and a decrease in Bacteroidetes Notably, the relative abundance of beneficial genera such as Faecalibacterium and Ruminococcaceae rose in the organic acids group, while the levels of Coprobacter, Lactobacillus, Bacteroides, and Lachnoclostridium diminished.
3.6.3 Impact of EOAs on intestinal microbiota
Supplementation with a blend of organic acids (OAs) and essential oils (EOs), specifically calcium butyrate, fumaric acid, citric acid, thymol, cinnamaldehyde, and carvacrol in a ratio of 1:8:1, significantly increased the relative abundance of beneficial gut bacteria, Ruminococcaceae and Lachnospiraceae.
The blend of essential oils, including thymol (5%), cinnamaldehyde (15%), citric acid (10%), sorbic acid (10%), malic acid (6.5%), and fumaric acid (13.5%), significantly increases the abundance of beneficial bacteria such as Firmicutes and Bacteroidetes at the phyla level, as well as Lactobacillus and Streptococcus at the species level, in weaned piglets This blend also effectively reduces the prevalence of harmful bacteria, specifically Enterobacteriaceae and Helicobacteraceae.
Impact of EOs, OAs and EOAs on function of intestinal microbiota
3.7.1 Impact of EOs on function of intestinal microbiota
Birds fed with 20% carvacrol and 25% thymol exhibited significant changes in their cecal microbiota, highlighting the presence of encoded enzyme genes in 15 pathways at a dose of 200 mg/kg and 20 pathways at 400 mg/kg These pathways included essential processes such as protein digestion and absorption, amino acid metabolism, lipid biosynthesis, lipopolysaccharide production, citrate cycling (TCA), and lipoic acid metabolism.
Chickens supplemented with essential oils (EOs), specifically thymol and carvacrol in a 25% + 25% ratio, showed a reduction in key processes within the KEGG groups, such as metabolic, genetic, and environmental information processing This included a decline in replication, recombination, and repair processes, as well as genes associated with biogenesis, ribosomal structure, and translation in the ileum microbiota Additionally, significant variability in the functional characterization of cecal microbiota composition was observed, particularly regarding the metabolism of carbohydrates and energy genes when utilizing bioactive phenolic compounds.
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35 extracts in broiler chickens [119] Metabolism of amino acids, biosynthesis of proteins, and metabolism of lipids were found to be enriched in the EOs treatment, according to metabolomics analysis [127]
4 Research Objective, Contents, Methods and Experimental route
Research Objective
Recent literature highlights the effectiveness of combining essential oils (EOs) and organic acidifiers (OAs) in promoting the health and growth of broilers, layers, and pigs Several studies demonstrate that these compounds facilitate the sustainable release of vegetable oils and organic acids, thereby enhancing broiler growth and overall health This research provides a solid theoretical foundation and technical support for the use of EOs and OAs in healthy broiler production Additionally, experiments indicate that EOs and OAs can positively influence the immune function of animals, although there is a limited number of studies exploring their mechanisms of action.
This research aims to address the gaps in existing studies regarding the synergistic and antagonistic effects of essential oils (EOs) when used individually or in mixtures The primary objective is to evaluate the impact of EOs and organic acids (OAs) on growth efficiency, intestinal integrity, immune response, and microbial metabolism in broiler chickens, while also exploring the mechanisms of their interactions.
The impact of essential oils (EOs) and organic acids (OAs) on the transformation of intestinal microorganisms in the digestive system remains poorly understood Therefore, further research is essential to explore the synergistic effects of combining EOs with OAs across the diverse range of intestinal microorganisms.
(2) In addition, nutritional physical metabolism and chemical metabolites may help us understand how dietary EOs and OAs affect intestinal metabolism
(3) Moreover, whether or not the intestinal barrier and its mechanism are regulated
The application of whole omics technology reveals the structural and functional dynamics of intestinal microflora, shedding light on the mechanisms behind the combined effects of essential oils and organic acids in promoting growth and health in broilers under stress.
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Contents and Methodology
This study aimed to evaluate the effectiveness of essential oils and organic acids in controlling subclinical necrotic enteritis (NE) and E coli challenges in broiler chickens, compared to antibiotic growth promoters (AGPs) and a control group without AGPs or probiotics Key metrics assessed included growth efficiency indicators such as body weight (BW), average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR) Additionally, the research examined intestinal morphological changes, gut lesion scores, cecal colonization by C perfringens or E coli, and the expression of their toxin genes The study also analyzed jejunal microbiota through 16S gene pyrosequencing to predict metabolic functions, alongside evaluations of intestinal permeability, barrier function, and immune responses induced by C perfringens or E coli in broilers.
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Experimental route
Dietary encapsulated essential oils and organic acids mixture improves gut health in broiler chickens challenged with necrotic enteritis
Evaluation of the blend of encapsulated essential oils and organic acids as antibiotic growth promoter alternative on growth performance and intestinal health in broilers challenged with necrotic enteritis
Effects of dietary EOA on growth performance, immunological parameters and gut microbiaota status of broiler chickens challenged with E.coli O78
The evaluation process includes assessing body weight (BW), average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR) Additionally, it involves analyzing intestinal lesion scores and the morphological structure of the intestine, along with the presence of inflammatory cells The assessment extends to evaluating bacterial levels in the intestine and liver, intestinal permeability through serum FITC-D detection, and the expression of immune-related genes in the intestinal mucosa Furthermore, the evaluation includes the expression of tight junction protein genes, microbial 16S RNA analysis, and metabolomic profiling.
Study on the mechanism of the mixture of EOs and OAs on the regulation of broiler performance and intestinal health
1 Effect of dosage of EOs and OAs in different breeding environments (infected with
2 The combination of EOs and OAs the regulation mechanism of chicken intestinal microflora and immunity
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Dietary encapsulated essential oils and organic acids mixture improves gut health
Introduction
The rapid increase in the global population has led to a significant rise in demand for animal protein, particularly in the poultry sector, where chicken production is expanding quickly This growth, however, raises health concerns due to the heightened risk of disease outbreaks on farms To combat pathogenic bacteria such as Clostridium perfringens, Escherichia coli, and Salmonella spp., antibiotics have been widely used for both prevention and treatment, enhancing disease control, production efficiency, and product quality Nevertheless, the frequent use of in-feed antibiotics to promote growth has come under scrutiny due to the potential development of antibiotic resistance among various bacterial strains.
The ongoing use of antibiotics disrupts the balance of sensitive bacteria in the gut, leading to an increase in drug-resistant microorganisms, particularly those resistant to antibiotic growth promoters (AGPs) This practice not only fosters the prevalence of these resistant strains but also contributes to therapeutic antimicrobial cross-resistance Research has highlighted the significant impact of AGPs on the rise and spread of antimicrobial-resistant bacteria, which prompted the European Union to ban their use in 2006 Additionally, South Korea became the first Asian country to prohibit AGPs in animal feed In July 2019, China's Ministry of Agriculture and Rural Affairs announced plans to ban all AGP feed additives, excluding traditional Chinese medicine, starting January 1, 2020.
In recent years, countries worldwide, particularly in Asia, have been working to reduce the use of antibiotics for growth promotion due to concerns about antibiotic resistance in livestock and its implications for public health The emergence of drug-resistant bacteria underscores the need for alternative strategies to manage infections caused by these pathogens Researchers have focused on alternatives to antibiotics, including essential oils, organic acids, probiotics, prebiotics, and minerals, to improve animal performance and health.
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In recent years, essential oil products have gained popularity due to their numerous advantages, including the lack of harmful residues and their widespread safety in the food industry Research has shown that essential oils possess significant potential to replace antibiotics, attributed to their antibacterial, anti-inflammatory, and antioxidant properties However, some studies indicate that dietary essential oil supplements may not significantly affect or enhance growth performance.
Organic acids, such as propionic, acetic, formic, and butyric acids, have long been utilized in feed preservation and as additives to improve broiler performance These simple monocarboxylic acids exhibit strong antimicrobial properties Additionally, salts like sodium butyrate have been shown to enhance growth performance in broilers and mitigate the effects of subclinical necrotic enteritis caused by C perfringens.
Despite the extensive use of essential oils and organic acids in various studies, the potential synergistic or antagonistic interactions between them have not been thoroughly examined within the same research framework Understanding these interactions is crucial for uncovering the mechanisms that can help prevent diseases impacting the poultry industry, particularly when combining essential oils and organic acids in diets as alternatives to antibiotic growth promoters (AGPs).
2 Basics overview of essential oils and acids
2.1 Basics overview of essential oils and their antibacterial mechanism
EOs are liquid aromatic fluid compounds from plants such as the leaves, stems, roots, flowers, seeds or fruits by steam distillation solvent extraction, steam distillation or extrusion
Essential oils are primarily composed of complex compounds, including terpenes and their oxygenated derivatives known as terpenoids, as well as aromatic and aliphatic acid esters and phenolic compounds According to Hammer and Carson, terpenoids are the predominant constituents found in essential oils, while phenyl-propanoids, although present, are less common.
Essential oils consist of 85% main ingredients, with minor components featuring 2 to 3 key ingredients that define their characteristics For instance, thymol and carvacrol, which comprise approximately 80% of oregano's essential oils, are significant phenols known for their antibacterial and antioxidant properties Additionally, oregano contains other compounds like r-cymene, which, while not as prominent, contribute to its overall profile.
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18 effective against bacteria, it creates favourable conditions for carvacrol transport through the cytoplasmic membrane [22]
Essential oils (EOs) possess unique chemical structures and biological effects influenced by various factors such as species, climate, ecology, harvesting time, and isolation methods Research indicates that approximately 3,000 plant families, including Alliaceae, Apiaceae, Asteraceae, Lamiaceae, Myrtaceae, Poaceae, and Rutaceae, are known for their medicinal value Notable plants with essential oils include black pepper (Piperaceae), garlic (Liliaceae), cinnamon (Lauraceae), anise (Apiaceae), thyme (Myrtaceae), turmeric (Zingiberaceae), and oregano.
Supplementing poultry and pigs with essential oils has been shown to modulate microbiota and enhance performance, leading to improved average daily gain (ADG) Research indicates that essential oils enhance feed digestion by increasing bile salt secretion and stimulating enzymatic activities in the pancreatic and intestinal mucosa Their antimicrobial properties, linked to their chemical structure, operate through a diverse range of mechanisms.
Essential oils, due to their hydrophobic properties, significantly impact cell membranes by puncturing the cell wall and altering the structure of the phospholipid bilayer in the cytoplasmic membrane This disruption increases the permeability of the cell membrane, resulting in the leakage of ions and protons, which ultimately affects the internal environment of bacterial cells Microscopic studies indicate that even at low concentrations, certain essential oils can create holes in the cell walls of sensitive bacteria, such as C perfringens, particularly in their vegetative and lysed forms.
Carvacrol disrupts the osmotic balance and alters the intracellular pH of bacteria by facilitating ion transport across the membrane The undissociated form of carvacrol penetrates the cytoplasmic membrane, acting like an H+ proton within the bacterial cytoplasm It then interacts with a K+ ion and exits the cell, carrying positive ions with it This process acidifies the cytoplasmic environment, impacting the membrane's proton pump mechanism and ultimately affecting ATP production within the cell.
(3) Changes in cellular metabolism: For example, thymol has been shown to disrupt citrate metabolism in Salmonella (reviewed by F Nazzaro) [36]
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Cinnamaldehyde, a compound found in cinnamon, inhibits the synthesis of contact molecules that bacteria use for communication, known as quorum sensing (QS) This inhibition limits the pathogenic properties of both Gram-positive (G+) and Gram-negative (G-) bacteria by reducing their growth, aggregation, biofilm formation, and the expression of various virulence factors Consequently, the use of essential oils (EOs) can effectively disrupt bacterial communication systems, thereby enhancing antibacterial activity and mitigating harmful microbial effects.
(5) Other mechanisms of essential oil might be linked to nutrient absorption inhibition, enzymatic inhibition, DNA, RNA, and protein synthesis by bacterial cells [31][38]
Figure 1 - 1 The essential oils' mechanism of action and target locations on microbial cells
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2.2 Basic overview of acidifiers and their antibacterial mechanisms
In addition to essential oils, various acids and their salts are increasingly being utilized as alternatives to antibiotics in animal feed, particularly in livestock and poultry These acids, including organic and fatty acids, exhibit antibacterial properties and promote growth Organic acids can be categorized based on the length of their carbon chains—short, medium, or long—or their saturation level, which can be either saturated or unsaturated Notably, some organic acids serve as intermediaries in the tricarboxylic acid (TCA) cycle, contributing to energy production for essential biological functions.
Some antimicrobial activity has been shown for different acidifiers and organic acids
Organic acids (OAs) added to animal and poultry feeds effectively reduce Salmonella levels due to their strong antibacterial properties For instance, benzoic acid has been shown to enhance growth efficiency in weanling piglets, resulting in a 12% increase in average daily gain (ADG) The antibacterial action of OAs involves lowering pH levels and their ability to penetrate bacterial cells, leading to a decrease in intracellular pH and inhibiting microbial metabolism This process can starve bacteria by disrupting their energy utilization and denaturing essential proteins and DNA Additionally, OAs reduce feed buffering capacity, which increases hydrochloric acid production in the stomach, thereby improving nutrient digestibility and stimulating the secretion of pancreatic enzymes, further enhancing overall nutrient absorption.
Materials and methods
2.1 Experimental design, birds and diets
A 2 × 2 completely randomized factorial design was implemented to investigate the effects of two levels of supplemental EOA (0 and 500 mg/kg of diet) alongside two degrees of NE challenge (challenged and unchallenged) in male broiler chicks A total of 288 one-day-old male broiler chicks were sourced from Beijing Arbor Acres Poultry Breeding Company and randomly assigned to four experimental groups, with six replicates of 12 birds each The treatment groups included: (i) a negative control group without EOA supplementation or NE infection; (ii) an EOA-treated group receiving 500 mg/kg EOA from days 1 to 42 without NE; (iii) a NE-infected control group without EOA supplementation; and (iv) an EOA-treated and NE-infected group receiving 500 mg/kg EOA while being challenged with NE.
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The study conducted by Menon Animal Nutrition Technology Co Ltd in Shanghai, China, involved raising non-infected and necrotic enteritis (NE) birds in separate areas to prevent cross-contamination Following the AA Broiler Management Guide, all birds were subjected to continuous light for the first 24 hours, transitioning to a 23-hour light and 1-hour dark schedule for the remainder of the experiment The temperature was maintained at 33°C–34°C during the initial three days post-hatching, gradually decreasing by 2°C weekly until reaching 22°C–24°C A pelleted basal diet, free from antibiotics and coccidiostats, was formulated in accordance with Arbor Acres broiler chicken nutrient specifications for both the starter (1–21 days) and grower (22–42 days) periods, with food and water provided ad libitum throughout the trial.
Birds infected with NE were performed as described previously with some changes
On day 14 post-hatch, chickens in the challenged treatments were orally gavaged with a mixture of Eimeria maxima (1.0×10^4 oocysts/bird) and Eimeria necatrix (5.0×10^3 oocysts/bird), sourced from Prof Suoxun at the College of Veterinary Medicine, China Agricultural University This was followed by an oral gavage of 1 mL containing C perfringens type A CVCC52 obtained from the China Veterinary Culture Collection Center.
Institute of Veterinary Drug Control, Beijing, China) at 2.2×10 8 colony-forming units (CFU)/mL per day from days 18–20 Similarly, birds in the unchallenged group were given
1 mL of sterile phosphate-buffered saline by oral gavage at the same time-points Feed was stopped 8 hours before each inoculation
On days 1, 21, and 42, body weight and feed consumption were measured for each replicate cage, leading to the calculation of body weight gain (BWG), average feed intake (AFI), and feed conversion ratios (FCRs).
2.4 Intestinal lesion scores and sample collection
At 7 days post infection (7DPI), after fasting, two birds/pen were randomly chosen and weighed individually, and then euthanized by jugular exsanguination When collecting
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In a study involving 42 samples, the intestines of each bird were longitudinally opened to assess necrotic enteritis (NE) lesions in the upper and lower intestines using a 0-4 scale evaluated by three independent observers Middle sections of the jejunum, approximately 1 cm in length, were washed with sterile saline, placed in sterile tubes, and immediately snap-frozen in liquid nitrogen for mRNA expression analysis at −80 °C Additionally, another jejunum sample of about 2 cm was rinsed in 0.9% physiological saline and preserved in 4% paraformaldehyde at 21 °C for morphological analysis Liver and cecal samples were also aseptically collected, snap-frozen in liquid nitrogen, stored at −80 °C, and transported for microbial culture and 16S rRNA analysis.
2.5 Histomorphological structure and goblet cell analysis of the jejunum
The morphological structure of the jejunum was analyzed using a standardized method, where jejunal samples were rinsed and fixed in a 4% buffered paraformaldehyde solution Following fixation, the samples underwent dehydration, clearing, and infiltration with paraffin, before being embedded in paraffin blocks Each block was sectioned into 5.0 µm thick slices, with two slices per block, and stained using the hematoxylin and eosin method Morphometric analyses were conducted using a Nikon phase-contrast microscope and a MicroComp digital imagery analysis system, measuring at least five villi and crypts per section across two sections per segment to determine villus height (VH) and crypt depth (CD), with averages calculated for statistical analysis Additionally, goblet cells in the jejunum were stained with Periodic Acid-Schiff (PAS), and the number of PAS-positive cells per villus was quantified using ImageJ software.
2.6 Microbiological measurements, intestinal permeability analysis by measuring microbial translocation and fluorescein isothiocyanate dextran (FITC-D) concentrations in serum
Approximately 1 g of the collected liver and cecal samples of each replicate were analyzed to determine the number of lactic acid bacteria, Coliform bacteria (faecal samples only) and Clostridium perfringens (both liver and feces samples) using conventional culture
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In this study, we employed 43 methods to select the appropriate medium for different microbial types, such as C perfringens on tryptose sulfite cycloserine (TSC) agar with cycloserine, E coli on Eosin-Methylene Blue agar, and Lactobacillus on MRS (de Man, Rogosa, Sharpe) agar Samples were homogenized and diluted from 10^-1 to 10^-7 using PBS solution under sterile conditions, followed by plating on selective agar to detect bacteria Traditional microbiological methods were used for incubation and calculating colony-forming units (CFUs) The plate-pouring technique specifically counted C perfringens cells in the liver, with bacterial translocation expressed in log10 CFU/gram of tissue.
At 7 DPI, 2 chickens each replicate were orally gavaged with FITC-D (3000–5000 Da molecular weight, Sigma Aldrich, St Louis, MO, USA) at 8.32 mg/mL/bird Samples of blood were taken at 1 and 2.5 h after administering FITC-D, then centrifuged at 3000 rpm 10 min to separate serum for FITC-D analysis as previously described [149] In brief, standard curves (0, 0.0001, 0.001, 0.01, 0.1, 1.0 and 10 àg/mL) were prepared using FITC-D Excitation and emission wavelengths of 485 nm and 528 nm, respectively, were used to test FITC-D concentrations in diluted sera (1:5 ratios) (Synergy HT, multi-mode microplate reader, BioTek Instruments, Inc., VT, USA) Based on the standard curve the FITC-D content per mL in serum had been measured
2.7 Real-time polymerase chain reaction (PCR)
Total RNA was isolated from approximately 60 mg of jejunum mucosa using Trizol reagent (Invitrogen Life Technologies) according to the manufacturer's instructions RNA quality was assessed via an agarose gel, while concentration and purity were measured with a Nanodrop-2000 spectrophotometer (Thermo Fisher Scientific) at 260 nm and 260/280 nm Subsequently, RNA was reverse transcribed into complementary DNA using the Primer Script™ RT reagent Kit (Takara Bio Inc) and stored at -20°C for future analysis The target gene sequences and primers for the reference gene are detailed in Tables 2-2 and 2-3, focusing on the expressions of TLR-2, TLR-4, MyD88, and TRAF.
The expression levels of key genes, including NF-kB, TNFSF15, IL-1β, IL-8, IFN-γ, IL-10, Tollip, PI3K, A20, SOCS-1, SOCS-6, as well as tight junction proteins and growth factors such as Claudin-1, Occludin, ZO-1, Mucin-2, TGF-β3, IGF-2, EGFR, and GLP-2, were quantified using SYBR Premix Ex TaqTM kits on the Applied Biosystems 7500 Fast Real-Time PCR System The specificity and efficiency of the primers were confirmed through melt curve analysis The 2 −ΔΔCT method was employed to calculate the mRNA expression levels of the target genes, with β-actin serving as the reference gene.
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2.8 16S rRNA amplification, sequencing and data processing of microbiota diagnostics
The microbial DNA genome from cecal samples of 28-day-old subjects was extracted using the QIAamp Fast DNA Stool Mini Kit (QIAGEN, Germany) The quantity and quality of the extracted DNA were assessed using a Nanodrop-2000 spectrophotometer, and its integrity was verified through agarose gel electrophoresis Qualified DNA served as a template for amplifying the microbial 16S rRNA V3-V4 gene region using barcoded primers F341 and R806 with the KAPA HiFi Hotstart Ready Mix PCR kit (Kapa Biosystems, USA) The PCR amplification was performed under specific conditions: an initial denaturation at 94°C for 5 minutes, followed by 30 cycles of denaturation at 95°C for 30 seconds, annealing at 50°C for 30 seconds, and extension at 72°C for 30 seconds, concluding with a final extension at 72°C for 5 minutes The resulting PCR products were analyzed using 2% agarose gel electrophoresis and purified with the QIAquick Gel Extraction Kit (QIAGEN, USA) Sequencing of the amplicon libraries was conducted on the Illumina MiSeq PE250 platform (Illumina, Santa Clara, CA, USA) using the MiSeq Reagent Kit in accordance with standard protocols (Shanghai Personal Biotechnology Co., Ltd., Shanghai, China).
The raw data obtained from the Illumina MiSeq platform (version 1.8.0-dev) were filtered and demultiplexed, with raw tags generated by merging sequence data using FLAST Forward and reverse sequences were combined after trimming and uploaded to QIIME for richness analysis The sequences were clustered and classified into reference OTUs (operational taxonomic units with 97% similarity) using UCLUST within QIIME Subsequently, MOTHUR software was employed to calculate α-diversity and β-diversity, with UniFrac distance differences assessed using the Student's t-test.
A 1000 permutation Monte Carlo test was conducted for pairwise comparisons of groups, with results visualized using box and whisker plots Principal component analysis was performed on genus-level compositional profiles To illustrate shared and unique operational taxonomic units (OTUs) among samples, a Venn diagram was created using R software, referencing the Silva taxonomic database Differences in microbial communities between experimental groups were assessed through partial least squares discriminant analysis (PLS-DA) using the "mix Omics" package Significant variations in microbial compositions between control and EOA-treated chickens were identified using a nonparametric Mann–Whitney U test, ranking the results by the percentage representation of individual genera.
Data regarding growth efficiency, intestinal lesion scores, intestinal bacterial concentrations, liver C perfringens, jejunum morphology, goblet cell numbers, intestinal
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The analysis of permeability, relative mRNA expression levels, and the Shannon and ACE alpha diversity indices among the four groups was performed using one-way ANOVA in SPSS 20.0, adhering to a 2 × 2 factorial design Duncan’s multiple comparison method was utilized for mean separations when significant interactive effects were observed Additionally, the Kruskal–Wallis test, along with Benjamini–Hochberg P-value correction, was applied to assess phylum and genus abundances A significance level of P ≤ 0.05 was established, while a P-value range of 0.05 ≤ P ≤ 0.10 was considered indicative of a tendency.
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Table 2 - 1 The composition and nutritional content in the experimental basal diet as well as when fed, except otherwise specified, %
1 VT (Vitamin) premix per kg complete dietary including: 30 IU vitamin E (DL-a-tocopherol acetate); 2500 IU vitamin
Results
Table 2 - 4 highlights the growth performance of broiler chickens, indicating that the untreated NE-challenged group experienced a significant reduction in body weight gain (BWG) from days 1 to 21 and 1 to 42, along with decreased average feed intake (AFI) during the first 21 days, and a notably increased feed conversion ratio (FCR) at various stages (P < 0.01) In contrast, dietary supplementation with essential oils (EOA) led to a significant improvement in FCR (P < 0.01) and a marked reduction in AFI (P < 0.05) during the later stages and overall trial period compared to the unsupplemented group.
During the period from days 22 to 42, a significant interaction was noted between feed conversion ratio (FCR) and average feed intake (AFI) in non-infected birds administered EOA These birds exhibited a substantial decrease in AFI (P ≤ 0.05) and a notable enhancement in FCR (P < 0.01) when compared to the NE-challenged control group and other treatments.
3.2 Intestinal lesion scores and morphological observations
NE infection led to a significant increase in jejunum crypt depth and small intestinal lesion scores, while reducing villus height and the VH/CD ratio in infected chickens compared to uninfected ones Chickens on EOA-supplemented diets exhibited higher villus height and VH/CD ratios, and infected birds receiving EOA had lower gut lesion scores and crypt depths at 7 DPI than untreated NE-infected birds The interaction between EOA supplementation and NE infection positively influenced small intestinal health, resulting in decreased lesion scores and crypt depths, along with an increased VH/CD ratio in NE-infected chickens on EOA diets However, there were no significant differences in jejunal goblet cell counts among the groups at day 28.
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3.3 Invasion of C perfringens into liver and FITC-D concentrations in serum
A significant interaction was observed between liver C perfringens invasion and cecal C perfringens colonization in NE-infected chickens supplemented with EOA The results indicated that NE-infected chickens on EOA-added diets showed a marked reduction in C perfringens levels.
C perfringens (P < 0.05) in the liver and cecal contents throughout the infection period compared to those in the NE-infected chickens The amount of C perfringens in the cecum and liver of the NE-infected birds at 7 DPI notably increased (P < 0.01) as compared with those of the uninfected chickens Conversely, the C perfringens community in the livers and ceca of the EOA-treated birds at 7 DPI declined dramatically (P < 0.01) as compared with those of the unsupplemented group The interaction between EOA supplementation and NE infection had a combined effect on the serum FITC-D concentration at 1 h post-FITC-D gavage (Table 6) Compared with NE-infected birds and the untreated groups, the infected and uninfected birds fed EOA exhibited lower serum FITC-D concentrations at 1 h post- FITC-D gavage (P < 0.05) however no notable impact was observed on the serum FITC-D concentration at 2.5 h post-FITC-D gavage
3.4 Expression of the intestinal tight junction and mucin-2 genes
Table 2-7 illustrates the effects of NE infection on tight junction, mucin-2, and growth factor mRNA expression in the jejunum NE infection significantly downregulated occludin, zonula occludens-1 (ZO-1), epithelial cell growth factor receptor (EGFR), and mucin-2 mRNA levels, while it notably upregulated GLP-2 and IGF-2 mRNA levels compared to unchallenged groups (P < 0.05) In contrast, EOA-treated birds exhibited lower ZO-1 and higher IGF-2 and GLP-2 expression levels than unsupplemented controls Furthermore, a significant interaction between NE infection and EOA supplementation affected claudin-1, IGF-2, and mucin-2 mRNA expressions Challenged chickens receiving EOA-supplemented diets demonstrated significantly higher claudin-1 and IGF-2 gene expression levels (P < 0.05) at 7 DPI in the jejunum compared to NE-infected birds Additionally, uninfected birds on EOA-supplemented diets showed the highest mucin-2 mRNA levels in the jejunum relative to the other treatment groups.
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3.5 mRNA levels of TLR signaling-related cytokines and growth factors in the jejunum
NE infection led to a significant decrease in the mRNA levels of TLR-4, TRAF-6, NF-κB, TNFSF15, TOLLIP, PI3K, and SOCS-6 in the jejunum, while markedly increasing the levels of IFN-γ mRNA compared to the unchallenged groups (P < 0.05).
2 - 8) The infected birds fed EOA exhibited lower levels of TLR-4 and TRAF-6 mRNA (P
< 0.05), raised levels of A20 mRNA and decreased IL-1β gene expression levels (0.05 < P
< 0.10) compared with the unsupplemented groups A dramatic interaction influence (P < 0.05) on TLR-2, TRAF-6, TNFSF15, TOLLIP and SOCS-6 mRNA levels occurred between
Chickens infected with necrotic enteritis (NE) and supplemented with essential oils (EOA) showed significantly reduced expression levels of TRAF-6, TNFSF15, and TOLLIP genes (P < 0.05) at 7 days post-infection (DPI) in the jejunum, alongside a decreasing trend in TLR-2 mRNA levels (0.05 < P < 0.10) compared to uninfected birds Conversely, uninfected birds receiving EOA exhibited the highest SOCS-6 gene expression levels in the jejunum at 7 DPI compared to the other groups.
This study investigated the impact of essential oil additives (EOA) on the gut microbiota of broiler chickens infected with necrotic enteritis (NE) by analyzing cecal microbiome contents through deep sequencing A total of 769,274 high-quality sequences were obtained from 24 samples, with an average of 45,685 reads per sample Using the Greengenes database and QIIME, operational taxonomic units (OTUs) at various taxonomic levels, including phylum and genus, were characterized A Venn diagram revealed 1,776 core OTUs shared among all groups, along with 209, 309, 382, and 235 unique OTUs for the four experimental groups Additionally, alpha diversity assessments using ACE, Chao1, Simpson, and Shannon indices indicated no significant changes in community richness and diversity of cecal feces (P > 0.05).
The study found that NE infection and EOA treatment, whether alone or in combination, did not significantly impact the alpha diversity of cecal microbial diversity Principal component analysis indicated a notable variability in cecal microbiota composition among the groups, while PLS-DA scores demonstrated a clear separation between untreated NE-infected birds and those receiving EOA treatment.
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This study examines the effects of EOA supplementation on the cecal microbial composition in birds challenged by necrotic enteritis (NE) We analyzed the differences in bacterial taxa between the A and B groups, as well as the D and G groups Our findings at the phylum level indicate that NE infection, EOA treatment, and their combined effects significantly influenced the proportions of Firmicutes.
On day 28, the relative abundance of Bacteroidetes was significantly affected (P < 0.05), while the levels of Proteobacteria, Actinobacteria, and other bacterial phyla remained unchanged Under unchallenged conditions, the addition of EOA led to a notable increase in Firmicutes abundance (P = 0.055) and a corresponding reduction in other bacterial groups.
In a study examining the effects of essential oils on poultry, Bacteroidetes levels were observed (P = 0.078) Among NE-challenged birds, those receiving essential oils (EOA) did not show significant differences in phyla relative abundances However, at the genus level, uninfected birds fed EOA displayed an increase in Lactobacillus (P = 0.081) and Coprococcus (P = 0.007), while Rikenellaceae levels decreased (P = 0.078) In contrast, NE-infected chickens treated with iEOA showed a trend towards increased relative abundance of Unclassified_Lachnospiraceae (P = 0.109) and a significant reduction in Erysipelotrichaceae levels (P = 0.031).
中国农业大学博士学位论文 Chapter 2
Table 2 - 4 Effect of EOA on the growth efficiency of bird infection with NE
Items Experimental design Main effect P-value 6
A 1 D 2 B 3 G 4 SEM 5 0 500 Non-Challenge Challenged Treatment Challenged T*C d 1 to 21
FCR, g/g 1.54 b 1.63 a 1.44 c 1.62 a 0.01 1.59 1.53 1.50 1.63