• Hyun Lillehoj, Yanhong Liu, Sergio Calsamiglia, Mariano E. Fernandez-Miyakawa, Fang Chi,Ron L. Cravens, Sungtaek Oh& Cyril G. Gay

Click to download the original PDf file!

Phytochemicals as antibiotic alternatives to promote growth and enhance host health

Abstract

There are heightened concerns globally on emerging drug-resistant superbugs and the lack of new antibiotics for treating human and animal diseases. For the agricultural industry, there is an urgent need to develop strategies to replace antibiotics for food-producing animals, especially poultry and livestock. The 2^nd International Symposium on Alternatives to Antibiotics was held at the World Organization for Animal Health in Paris, France, December 12–15, 2016 to discuss recent scientific developments on strategic antibiotic-free management plans, to evaluate regional differences in policies regarding the reduction of antibiotics in animal agriculture and to develop antibiotic alternatives to combat the global increase in antibiotic resistance. More than 270 participants from academia, government research institutions, regulatory agencies, and private animal industries from >25 different countries came together to discuss recent research and promising novel technologies that could provide alternatives to antibiotics for use in animal health and production; assess challenges associated with their commercialization; and devise actionable strategies to facilitate the development of alternatives to antibiotic growth promoters (AGPs) without hampering animal production. The 3-day meeting consisted of four scientific sessions including vaccines, microbial products, phytochemicals, immune-related products, and innovative drugs, chemicals and enzymes, followed by the last session on regulation and funding. Each session was followed by an expert panel discussion that included industry representatives and session speakers. The session on phytochemicals included talks describing recent research achievements, with examples of successful agricultural use of various phytochemicals as antibiotic alternatives and their mode of action in major agricultural animals (poultry, swine and ruminants). Scientists from industry and academia and government research institutes shared their experience in developing and applying potential antibiotic-alternative phytochemicals commercially to reduce AGPs and to develop a sustainable animal production system in the absence of antibiotics.

Introduction

Antibiotics, since their discovery in the 1920s, have played a critical role in contributing to the economic effectiveness of animal production as feed supplements at sub-therapeutic doses, to improve growth and feed conversion efficiency, and to prevent infections [1]. In-feed antibiotics (IFAs) are a common and well-established practice in the animal industry that has contributed to the intensification of modern-day livestock production. However, with intensification of animal agriculture, concerns exist that the use of IFAs leads to development of antimicrobial resistance, posing a potential threat to human health [2]. Although mixed opinions still exist on the transfer of antibiotic resistance genes from animal pathogens to those of humans, studies have shown a potential link between the practice of using sub-therapeutic doses of antibiotics and the development of antimicrobial resistance among the microbiota. In the US, antibiotic use in livestock and poultry feeds is under scrutiny as a result of increasing consumer awareness and the demand for livestock products from antibiotic-free production systems. In 2013, the US Food and Drug Administration (FDA) called for major manufacturers of medically important animal drugs to voluntarily stop labeling them for animal growth promotion [3], and published its final rule of the Veterinary Feed Directive (VFD) in 2015. The quest for alternative products has clearly intensified in recent years with the increase in regulations regarding the use of antibiotic growth promoters (AGPs) and the rise in consumer demand for poultry products from “Raised Without Antibiotics” or “No Antibiotics Ever” flocks [2]. There has been a significant increase in scientific papers in the recent literature on antibiotic alternatives and feed additives to promote growth and enhance gut health, and reduce the use of antibiotics in animal production. The classes of antibiotic alternatives that are available to increase animal productivity and help poultry and pigs perform to their genetic potential under existing commercial conditions include probiotics, organic acids, phytogenics, prebiotics, synbiotics, enzymes, antimicrobial peptides, hyperimmune egg antibodies, bacteriophages, clay and metals [2]. Although the beneficial effects of many of the alternatives developed have been well demonstrated, there is a lack of information on their mechanism of action, efficacy, and advantages and disadvantages of their applications in the field. Furthermore, the general consensus is that these products lack consistency and their efficacies vary among farms and locations. Therefore, their modes of action need to be better defined. Optimal combinations of various alternatives coupled with good management and husbandry practices will be the key to maximize performance and maintain animal productivity while we move forward, with the ultimate goal of reducing antibiotic use in the animal industry. With declining AGPs usage and increasing consumers’ concerns about superbugs, the quest for novel alternate replacements to mitigate antibiotic use in animal agriculture will grow significantly in the coming years. In this Phytochemical Session, we reviewed scientific evidence that phytochemicals stimulate innate immune cells, reduce oxidative stress, maintain gut integrity, promote beneficial bacteria growth, and reduce the negative consequences of inflammation caused by enteric infections as effective antibiotic alternatives to promote animal growth performance in poultry, swine, and beef and dairy production.

Plant-derived phytochemicals as antibiotic alternatives

Phytochemicals, also referred to as phytobiotics or phytogenics, are natural bioactive compounds that are derived from plants and incorporated into animal feed to enhance productivity [2]. Ideal antibiotic alternatives should have the same beneficial effects of AGPs, ensure optimum animal performance, and increase nutrient availability. Considering the proposed mechanism of action of AGPs in modulating the gut microbiome and immunity, a practical alternative should exert a positive impact on feed conversion and/or growth [2, 4]. Phytochemicals can be used in solid, dried and ground form or as extracts (crude or concentrated), and also can be classified as essential oils (EOs; volatile lipophilic substances obtained by cold extraction or steam/alcohol distillation) and oleoresins (extracts derived by non-aqueous solvents) depending on the process used to derive the active ingredients [2]. The main bioactive compounds of the phytochemicals are polyphenols, and their composition and concentration vary according to the plant, parts of the plant, geographical origin, harvesting season, environmental factors, storage conditions, and processing techniques [2]. In recent years, phytochemicals have been used as natural growth promoters in the ruminants, swine and poultry industries. A wide variety of herbs and spices (e.g., thyme, oregano, rosemary, marjoram, yarrow, garlic, ginger, green tea, black cumin, coriander and cinnamon) have been used in poultry for their potential application as AGP alternatives [2]. In contrast, several other phytochemicals such as grape pomace, cranberry fruit extract, Macleaya cordata extract, garlic powder, grape seed extract, and yucca extract, when tested as growth promoters, did not show any effects on performance parameters [2]. In addition to herbs and spices, various EOs (thymol, carvacrol, cinnamaldehyde, and eugenol, coriander, star anise, ginger, garlic, rosemary, turmeric, basil, caraway, lemon and sage) have been used individually or as blends to improve animal health and performance [2]. Variable results have been reported with the use of EOs in poultry diets, some including cinnamaldehyde [5,6,7], and a blend of thymol and cinnamaldehyde improved body weight gain in broilers, while others like thymol and EOs from star anise improved feed efficiency, as seen by reduced feed conversion ratio (FCR). Curcuma alone or curcuma with capsicum [7, 8] enhanced resistance to enteric diseases such as coccidiosis and necrotic enteritis. The variation in the results could be attributed to differences in the composition, type and origin of the EOs that were used, inclusion level, and the environmental conditions of the trials [2]. Nevertheless, one commercial blend of phytonutrients (containing carvacrol, cinnamaldehyde and Capsicum oleoresin), which enhances innate immunity and reduces negative effects of enteric pathogens [9, 10], was approved in the EU as the first botanical feed additive for improving performance in broilers and livestock. Several trials performed with this commercial blend have demonstrated consistent improvement in growth and feed efficiency [9,10,11]. A meta-analysis of 13 broiler studies involving the use of this commercial blend showed that its inclusion in diets increased body weight gain and decreased feed conversion ratio and mortality [12]. The mechanism of action of phytochemicals is not clearly understood but may depend upon the composition of the active ingredients in the product being used. The beneficial effects of phytochemicals are attributed to their antimicrobial and antioxidant properties. In addition, the inclusion of phytochemicals in the diets alters and stabilizes the intestinal microbiota and reduces microbial toxic metabolites in the gut, owing to their direct antimicrobial properties on various pathogenic bacteria, which results in relief from intestinal challenge and immune stress, thus improving performance [13]. Another important beneficial effect of dietary inclusion of phytochemicals is reduction in oxidative stress and increase in antioxidant activity in various tissues, and thus, improved health [14]. Phytochemicals also exert their action through immunomodulatory effects such as increased proliferation of immune cells, modulation of cytokines, and increased antibody titers [5,6,7,8, 15,16,17,18]. In addition, phytochemicals in Allium hookeri improved gut barrier function, as demonstrated by increased expression of gut tight junction proteins in the mucosa of lipopolysaccharide (LPS)-treated young broiler chickens [18].

Examples of phytochemical antibiotic alternatives in poultry and livestock production

Dietary phytochemicals enhancing innate immunity in poultry

A growing body of scientific evidence has demonstrated that many of the health-promoting activities of phytochemicals are mediated through their ability to enhance host defense against microbial infections [4, 19]. The immune-activating properties of medicinal plants such as dandelion (Taraxacum officinale), mustard (Brassica juncea) and safflower (Carthamus tinctorius) have been evaluated in vitro using avian lymphocytes and macrophages [9]. All three extracts inhibit tumor cell growth, stimulate innate immunity and exert antioxidant effects in poultry [9]. Beneficial effects of cinnamaldehyde ((2E)-3-phenylprop-2-enal), a constituent of cinnamon (Cinnamomum cassia), a widely used flavoring compound that has been traditionally used to treat human diseases, has been investigated. Cinnamaldehyde stimulated primary chicken spleen lymphocyte proliferation in vitro and activated macrophages to produce high nitric oxide (NO) [6, 9]. Because of increased regulation of AGPs in poultry production, control of enteric diseases such as necrotic enteritis (NE) and coccidiosis, which have been traditionally controlled by in-feed antibiotics [2], needs antibiotic-free disease control strategies. Although plant-derived chemicals with potent medicinal properties are currently in clinical trials for treatment of a variety of diseases in humans, only limited research has documented the beneficial effects of phytochemicals on avian diseases [4, 19]. Dietary supplementation of 1-day-old chickens with cinnamaldehyde at 14.4 mg/kg showed up to 47-fold greater levels of gene transcripts encoding interleukin (IL)-1β, IL-6, IL-15 and interferon (IFN)-γ in intestinal lymphocytes, compared with chickens given a standard diet [15, 19]. Cinnamaldehyde-fed chickens showed 17 and 42% increased body weight gains following Eimeria acervulina and E. maxima infections, respectively, 40% reduced E. acervulina oocyst shedding, and 2.2-fold higher E. tenella-stimulated parasite antibody responses, compared with the control. The most reliable genetic network induced by dietary cinnamaldehyde treatment is related to antigen presentation, humoral immunity, and inflammatory disease. Chickens continuously fed 15 mg/kg anethole from hatch and orally challenged with live E. acervulina oocysts showed increased body weight gain, decreased fecal oocyst excretion, and greater anti-parasite serum antibody responses, compared with the control group. Global gene expression analysis by microarray hybridization in the intestinal lymphocytes of anethole-fed birds showed that many genes related to the inflammatory response are altered [17]. The levels of transcripts encoding IL-6, IL-8, IL-10 and TNF superfamily member 15 (TNFSF15) in intestinal lymphocytes were increased in parasite-infected chickens given the anethole-containing diet, compared with the control chickens given a standard diet. Garlic metabolites also have been tested in poultry using propyl thiosulfinate (PTS) and propyl thiosulfinate oxide (PTSO) [16]. Supplementation of 10 mg/kg PTS/PTSO increased body weight gain and serum antibody titers against profilin, an immunogenic protein of Eimeria, and decreased fecal oocyst excretion in E. acervulina-challenged chickens compared with chickens fed a control diet [16]. The addition of PTS/PTSO in broiler’s diet altered many genes related to innate immunity, including TLR3, TLR5 and NF-κB [16] and down-regulated expression of IL-10 compared with the control diet. In uninfected chickens, dietary supplementation with PTS/PTSO increased the levels of transcripts encoding IFN-γ, IL-4, and an antioxidant enzyme, paraoxonase 2, but decreased transcripts for peroxiredoxin-6 [16]. Combination of multiple phytochemicals exert synergistic effects to reduce negative consequences of enteric infections. Dietary supplementation of newly hatched broiler chickens with a mixture of Curcuma longa, Capsicum annuum (pepper), and Lentinus edodes improved body weight gain and serum antibody titers against profilin, and reduced fecal oocyst shedding in E. acervulina-infected birds, compared with the birds fed the control diet or a diet containing Capsicum plus Lentinus [5]. The effects of carvacrol, cinnamaldehyde and Capsicum oleoresin on the regulation of expression of genes associated with immunology, physiology, and metabolism have been investigated in chickens using high-throughput microarray analysis [15]. The levels of transcripts for IL-1β, IL-6, IL-15 and IFN-γ in gut lymphocytes were also greater in the Curcuma/Capsicum/Lentinus-fed birds, compared with those fed the standard, Curcuma or Capsicum/Lentinus diet. In a follow-up study, a combination of carvacrol, cinnamaldehyde and Capsicum oleoresin, or a mixture of Capsicum and Curcuma oleoresins increased protective immunity against experimental E. tenella infection following immunization with profilin, compared with untreated and immunized controls [10]. Immunized chickens fed the carvacrol/cinnamaldehyde/Capsicum-supplemented diet showed increased numbers of macrophages in the intestine, while those given the Capsicum/Curcuma oleoresin-supplemented diet had increased numbers of intestinal T cells, compared with untreated controls. While numerous studies have shown disease prevention or immune-enhancing effects of phytochemicals, few have examined the underlying mechanisms that are involved. Some phytochemicals inhibit innate immune response by targeting pathogen pattern recognition receptors or their downstream signaling molecules [20]. The Clostridium-related poultry disease such as NE causes substantial economic losses on a global scale [21]. It has been suggested that dietary phytonutrients could be used against NE. Supplementation of a mixture of Capsicum and Curcuma longa oleoresins (XTRACT^®) from hatch increased body weight and reduced gut lesion scores in NE-afflicted birds, compared with infected birds given the non-supplemented diet [7]. The XTRACT^®-fed birds also had lower serum α-toxin levels and reduced mRNA expression of IL-8, lipopolysaccharide-induced TNF factor (LITAF), IL-17A and IL-17F in intestine, but increased cytokine/chemokine levels in splenocytes, compared with birds fed with the control diet. This study documented the molecular and cellular immunity changes following dietary supplementation with extracts of Capsicum and turmeric that may be relevant to protective immunity against avian NE [7]. Future studies are needed to define the molecular and cellular mode of action of this phytochemical combination for the control of NE in the field.

Dietary phytochemicals on weaning pig health

Phytochemicals have been used for human nutrition and health improvement due to their potential biological functions, such as, antiviral, antimicrobial, antioxidant and anti-inflammatory effects [2, 5, 22]. Various phytochemicals exhibit a wide spectrum of antibacterial activities against Gram-negative and Gram-positive bacteria [23] with several different modes of action. First, phytochemicals directly kill bacteria due to their hydrophobicity, which enables them to partition into the lipids of the bacterial cell membrane and mitochondria, resulting in leakage of critical intracellular materials [24]. Second, phytochemicals contain a high percentage of phenolic compounds, which possess strong antibacterial properties [25]. Third, the active components in phytochemicals disturb the enzyme system of bacteria and block their virulence [26]. Fourth, certain bioactive components in phytochemicals may prevent the development of virulence structures in bacteria, such as flagella, which critical for bacterial adhesion [27]. Phytochemicals are also proposed for use as antioxidants in animal feed, which will protect animals from oxidative damage caused by free radicals. The antioxidative properties of extracts of oregano, thyme, clove, pepper, lavender and basil have been evaluated by many studies in vitro [28, 29]. Our recent in vitro assays have also revealed that EOs extracted from peppermint and spearmint have cellular antioxidant activities by increasing intracellular glutathione concentration in H₂O₂-stimulated intestinal epithelial cells (unpublished data). Frankič et al. [30] showed that supplementation of phytochemicals to pigs reduced DNA damage in lymphocytes, which indicates their potentially beneficial effects on the immune system under dietary-induced oxidative stress. The antioxidant activity of phytochemicals is highly correlated with their chemical composition [31]. Phenolic OH groups in thymol, carvacrol and other phytochemicals act as hydrogen donors to the peroxy radicals produced during the first step in lipid oxidation, thus retarding H₂O₂ formation [32]. The anti-inflammatory effects of phytochemicals have been widely reported in in vitro cell culture models. EOs from clove, tea, garlic, cinnamon and others have potential anti-inflammatory activities and suppress the production of TNF-α, IL-1β and NO from LPS-induced mouse macrophages [33]. Our previous research in vitro with porcine alveolar macrophages showed that carvacrol, Capsicum oleoresin, cinnamaldehyde, garlic, eugenol, anethol, and turmeric oleoresin suppress the production of proinflammatory cytokines (TNF-α and IL-1β) from LPS-stimulated macrophages [22], which indicates that all of these phytochemicals have anti-inflammatory effects. The modes of action for the anti-inflammatory activities of phytochemicals are not clear, but evidence suggests that these effects are partially mediated by blocking the nuclear factor (NF)-κB activation pathway [34]. For example, curcumin can block cytokine-induced NF-κB DNA binding activity, RelA nuclear translocation, IκBα degradation, IκB serine 32 phosphorylation, and IκB kinase activity [34]. Weaning is one of the most challenging and critical stages in swine production. Its effects are multifactorial, including behavior, environment, disease, immunity and nutrition. In this period, piglets are immediately subjected to a combination of stressors that predispose them to diarrhea, which can adversely affect survival at an early and most vulnerable stage [35]. The beneficial effects of phytochemicals on weaning pigs have been reported by different research groups. Manzanilla et al. [36] and Nofrarías et al. [37] have suggested that phytochemicals improve gut health. They have reported that a mixture of phytochemicals (XTRACT^®) standardized to 5% (w/w) carvacrol, 3% cinnamaldehyde, and 2% Capsicum oleoresin (oregano, cinnamon and Mexican pepper) increases stomach contents, suggesting an increased gastric retention time. In addition, XTRACT^® decreases ileal total microbial mass and increases the lactobacilli:enterobacteria ratio. Michiels et al. [38] have also indicated that supplementing with 500 ppm carvacrol and thymol reduces the number of intraepithelial lymphocytes and increases villus height/crypt depth in the distal small intestine. Escherichia coli post-weaning diarrhea is a common cause of death in weaned pigs. This diarrhea is responsible for economic losses due to mortality, morbidity, decreased growth performance, and cost of medication [39]. Enterotoxigenic E. coli are the most dominant types of pathogenic E. coli that cause diarrhea in both pre- and post-weaning piglets [40]. Capsicum oleoresin, garlicon, and turmeric oleoresin have been tested in an in vivo pathogenic E. coli challenge study to determine the effects of individual phytochemicals on diarrhea and gut health of weaning pigs [41]. The pigs were weaned at 21 days of age, transported to the experimental facility, and given the experimental diets immediately. After a 5-day adaptation period, they were challenged with three consecutive daily doses of 10¹⁰ colony forming units/3 mL of a hemolytic E. coli with F18 fimbria. The experimental diets were a control diet based on corn and soybean meal and three additional diets containing 10 mg/kg of each plant extract. The E. coli infection increased diarrhea score, frequency of diarrhea, and reduced growth rate, feed efficiency and villus height of the small intestine. However, supplementation with individual phytochemicals reduced overall frequency of diarrhea of pigs, indicating that feeding phytochemicals may enhance disease resistance in pigs. Supplementation with phytochemicals also improved ileal villus height and upregulated mRNA expression of the MUC–2 gene, which indicated that the reduced diarrhea score was likely due to improved gut barrier function and integrity. Pigs infected with E. coli showed an increased number of white blood cells, serum proinflammatory cytokine (TNF-α) and acute phase protein (haptoglobin) and increased recruitment of macrophages and neutrophils in the ileum. Dietary supplementation with phytochemicals reduced white blood cells, neutrophils, serum TNF-α and haptoglobin and the numbers of macrophages and neutrophils in the ileum compared with the control diet. These observations indicate that feeding low doses of phytochemicals reduces both systemic and local inflammation caused by E. coli infection. To decipher the underlying mechanism behind the benefits of feeding phytochemicals, microarray analysis has been conducted to characterize gene expression in the ileal mucosa of pigs experimentally infected with E. coli. Microarray results indicate that feeding phytochemicals enhances the integrity of membranes, especially several tight junction proteins. Supplementation of phytochemicals downregulates expression of genes related to antigen processing and presentation and other immune-response-related pathways, indicating that these phytochemicals attenuate the immune responses caused by E. coli infection [42]. Another in vivo study on porcine reproductive and respiratory syndrome virus (PRRSV) [43] showed that feeding Capsicum oleoresin, garlicon, and turmeric oleoresin to weaning pigs enhances the immune responses to PRRSV challenge and may help alleviate the negative impact of infection, as indicated by reduced viral load and serum concentrations of inflammatory mediators, and shortened duration of fever. In summary, phytochemicals are strong candidates to replace antibiotics to improve growth performance and health of pigs. The potential benefits of plant extracts may differ due to the large variation in the composition of plant extracts. This diversity prompts us to select optimal feed additives for evaluating their possible roles as alternatives to antibiotics in swine production.

Use of phytonutrients in ruminants

In ruminants, the host and rumen microorganisms establish a symbiotic relationship by which the animal provides nutrients and the proper fermentation conditions, and microbes degrade fiber and synthesize microbial protein as an energy and protein supply for the host, respectively. Carbohydrates are fermented in the rumen into pyruvate, resulting in the production of metabolic hydrogen. Volatile fatty acids (VFAs) are natural hydrogen sinks that help maintain the equilibrium of hydrogen and the fermentation process active. Retention of energy from glucose is the highest in propionate (109%), intermediate in butyrate (78%) and the lowest in acetate (62.5%). Although methane is effective in retaining hydrogen, the energy retained is lost through eructation and not available to the host. Manipulation of the relative proportions of these VFAs is key to the development of targets to modify rumen microbial fermentation [44]. Protein degradation is also important for the supply of nitrogen to rumen microbes for their growth, but excess ammonia nitrogen is absorbed through the rumen wall, transformed into urea in the liver, and excreted through the urine. In most production systems, ammonia nitrogen in the rumen is produced in excess of the ability of rumen microbes to use it, resulting in significant production costs and an increase in the release of nitrogen into the environment [45]. Therefore, controlling proteolysis, petidelysis and deamination should also be considered targets of interest in the modulation of rumen fermentation [44]. In fact, in a recent study, Van der Aar et al. [46] indicated that improving the efficiency of the digestion processes in ruminants is still the most efficient strategy to improve animal performance. AGPs are efficient in shifting rumen fermentation towards more efficient energy and nitrogen utilization pathways [47], improving productivity in dairy and beef diets [48, 49]. Therefore, industry is searching for alternative feeding strategies and/or additives that will allow it to maintain the current level of production without increasing the cost. Phytonutrients are a group of small organic molecules present in plants that modify the nutritional value of feeds by either modulating the digestion of nutrients in the digestive tract, or other systemic metabolic pathways. Some phytonutrients have a strong antimicrobial activity [50]. However, these molecules are not suitable for use in ruminants because the activity of rumen bacteria is essential for the proper function of the rumen. Research on alternatives to antibiotics as feed supplements in cattle should focus on molecules and doses that are able to produce subtle changes in the microbial metabolism and modify their rate of growth [51]. In the context of the continuous flow in the rumen, a change in growth rates results in changes in the proportion of rumen bacteria populations, resulting in changes in the fermentation profile. For example, Patra and Yu [52] were able to prove how different phytonutrients have different capacities in modifying the structure of the microbial population of the rumen. These changes are large in oregano (where thymol and carvacrol are the main active components) and peppermint (where menthol and menthone are the main active components) oils, but smaller, and more adequate, in clove bud (where eugenol is the major active component) and garlic oils. Ferme et al. [53] also have demonstrated that the reduction in protein degradation and ammonia production is achieved through changes in the total amount of Prevotella ssp. in the rumen; a major group of bacteria involved in amino acid deamination. These findings are important to set clear objectives in the search for alternatives to AGPs, which should identify phytonutrients that can modify the VFA proportions and protein degradation in the rumen without affecting nutrient degradation and the normal function of the rumen. Most phytonutrients of interest in animal feeding are classified into three main groups: saponins, tannins (饲用单宁酸) and EOs. Saponins and sarsaponins are the main active components of several phytochemicals, including yucca, quillaja, alfalfa and fenugreek. Saponins exhibit antibacterial [54] and antiprotozoal [54, 55] activity, resulting in a reduction in ammonia nitrogen concentration. Tannins (饲用单宁酸) are phenolic compounds found in almost every plant part, and are divided into two groups, hydrolysable and condensed tannins (饲用单宁酸). Condensed tannins (饲用单宁酸) have the ability to bind and precipitate proteins and may be useful in the control of protein utilization by ruminants [56], but at high levels may interfere with dry matter (DM) intake and digestibility of nutrients [56], and may decrease the incidence of bloating [55]. EOs are secondary plant metabolites present in many plants and may have a wide range of effects. In this section, we review recent research on the use of EOs as feed additives in ruminants.

Essential oils as modifiers of rumen fermentation

The increased rumen fermentation is indicated by the increase in propionate and decrease in methane, acetate and ammonia nitrogen, without reducing total VFA [57] in the in vitro fermentation system. When phytochemicals are tested, a considerable variation in fermentation with different extracts is observed due to the content of active compounds in these extracts [58]. Therefore, it is necessary to either report the concentration of these active compounds in phytochemicals, or use the active components to define activities, doses and mechanisms of action in an unequivocal form. For example, garlic oil reduces the proportions of acetate and branched-chain VFAs, and increases the proportions of propionate and butyrate in vitro [57, 59], and the fermentation profile is consistent with changes observed when methane inhibitors are supplied to ruminants. The anti-methanogenic effect of garlic and its active components is the result of direct inhibition of Archea microorganisms in the rumen through the inhibition of hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase; a specific pathway essential for the membrane stability of Archea [57, 59]. This observation was supported by Miller and Wolin [60], who reported similar effects when using statins, known to inhibit HMG-CoA reductase. However, benefits are often inconsistent, and strong inhibition of VFA production by garlic oil has been reported in some cases [59, 61, 62]. The variable effects of garlic oil on total VFA production is likely due to the short margin of safety in the doses between adequate and toxic levels. Cinnamaldehyde and eugenol also reduce the molar proportion of acetate, and increase the molar proportions of propionate and butyrate [59, 61]. These observations are consistent with improved energy retention by those phytochemicals and potentially due to the inhibition of methanogenesis [63]. Cinnamaldehyde also reduces ammonia nitrogen and increases free amino acids, suggesting that deamination of amino acids is inhibited in the rumen [59, 61]. Ferme et al. [53] have reported that cinnamaldehyde reduces Prevotella spp., bacteria involved in deamination, in an in vitro rumen simulation system. However, Eugenol inhibits the breakdown of large peptides to amino acids and small peptides [59]. The combination of eugenol and cinnamaldehyde may work in synergy to inhibit peptidolysis and deamination, and then improve the overall supply of amino acids and small peptides to microorganisms and the host. Therefore, a synergetic advantage could be expected by combining specific phytonutrients that work at different levels in the same metabolic pathway. There are limited data reported about the effects of phytochemicals on performance of ruminants. Feeding cinnamaldehyde alone or in combination with eugenol results in increased in milk production of 1.7–2.7% [64]. An even better response is reported when a combination of cinnamaldehyde, eugenol and capsicum is fed to dairy cattle, with increases in energy-corrected milk production of 5.2% [65] and 3.2% [66]. However, no differences have been observed in most of cases due to the small size of the studies. Bravo et al. [67] have summarized a large set of in vivo field trials using combinations of cinnamadehyde and eugenol through a meta-analysis, and have reported an improvement in milk production of 3.0% for dairy cattle.

Essential oils as modifiers of metabolic activities

Many phytonutrients have metabolic effects that are not related to their activities in the rumen [68, 69]. Preliminary in vitro rumen fermentation studies in dairy cattle have not identified capsicum as a potential modifier for rumen function [61, 70]. Capsicum increases DM and water intake in beef cattle from 9.2 to 14% [70,71,72], while these effects are not observed in dairy cattle [73, 74]. The benefits may be more significant when intake is compromised, such as when the cattle arrive at feedlots or during heat stress. The increase in DM intake patterns is probably also related to a more stable rumen pH [75]. Capsicum has been reported to modulate immune function [42]. Oh et al. [76] have reported an improvement in immunity indicators, with an increase in neutrophils and decrease in lymphocytes when cattle are fed rumen-protected capsicum. Feeding rumen-protected capsicum is reported to improve milk production. Stelwagen et al. [77] and Wall et al. [78] have reported increases in milk production of 6.6 and 9.1% in pasture and intensive production systems, respectively. Another three studies have also reported that supplementation of rumen-protected capsicum improved milk production by 6.2% [76], 10% [79], and 4.4% [80], respectively. The average increase in milk production in those studies was higher than the effects attributed to the modulation of rumen fermentation. Oh et al. [80] observed that supplementation with rumen-protected capsicum resulted in a lower insulin concentration after a glucose tolerance test. These results suggest that capsicum modifies glucose metabolism, redirecting glucose away from peripheral tissues and towards the mammary gland to increase milk production. In fact, Bovine somatotropin (bST) increases milk production by an average of 13%, redirecting glucose to the mammary gland, although the mechanism of action is different. This is an exciting new application of phytonutrients that presents an opportunity to improve production, not only by reducing the use of antibiotics, but also by providing an alternative to the use of some hormones. The average effect of rumen modifiers like monensin, yeast or some phytonutrients, commonly increase milk production by 2–4%, while capsicum increases milk production by an average of 7%.

Phytochemicals and the digestive microbiota

The mammalian gastrointestinal tract harbors a dense and diverse microbial community, which is composed primarily of bacteria but also includes fungi, Archaea and viruses. Collectively, these are referred to as intestinal microbiota. These microorganisms are environmentally acquired, and their metabolic functions can shape host physiology. Many vertebrates consume a diet rich in complex nutrients that are indigestible by their own intestinal enzymes, relying on the diverse biochemical catabolic activities of the microbiota. Available evidence strongly suggests that the gut microbiota plays important roles in host energy harvest, storage and expenditure, as well as overall nutritional status [81,82,83,84]. It must be highlighted that germ-free animals that lack any microbiota weigh less and have less fat than conventional animals [85], pointing out a key role of the microbiota in weight gain. Gut microbiota may affect weight gain through regulating nutrient extraction, and modulating the immune system and metabolic signaling pathways [82]. Many classes of substances with antibiotic activity that are effective for animal growth promotion display multiple modes of action and spectra of activity over the gastrointestinal microbiota. It has been difficult to predict which microbial changes are responsible for increases in weight gain, feed efficiency or health promotion. Culture-independent approaches using next-generation DNA sequencing have provided researchers with a revolutionary tool to look into microbiomes that could not be achieved before, and has begun to transform our view of intestine-associated biodiversity of animal production. Improving the understanding of microbiota and host metabolism would help to develop better strategies and products for animal production and welfare, food safety and public health. The selection of microbes that aid in nutrient extraction, regulating microbial carbohydrate, protein and lipid metabolism, and the prevention of subclinical infections will help to promote productive parameters [83]. The intestinal microbiota plays a critical role in inflammatory gut diseases of humans and animals [86]. Recent development and application of next-generation sequencing technologies using 16S rRNA gene have allowed investigation of the significant roles of the microbiota in gastrointestinal tract diseases, and have facilitated investigation of host–pathogen interaction in NE [86]. The effect of dietary phytochemicals on gut microbiota was studied in three major commercial broiler chickens fed with Capsicum and C. longa oleoresins [13]. Among the three chicken breeds, Cobb, Hubbard and Ross, oleoresin supplementation was associated with altered intestinal microbiota. The results suggested that dietary feeding of Capsicum and C. longa oleoresins reduces the negative consequences of NE, in part, through alteration of the gut microbiome. Although these are preliminary characterizations of the effects of dietary phytochemicals on gut microbiota but document the role of dietary Capsicum and C. longa oleoresins in regulating disease susceptibility to NE via altering the intestinal microbiota in commercial broiler chickens. A recent study [13] showed that Firmicutes was the dominant phylum and Lactobacillus was the predominant genus identified in the ileum in all broiler breeds and all treatment groups. These results are consistent with previous studies that showed Lactobacillus as the principal microorganism in the gastrointestinal tract of uninfected conventional broilers [87]. Because Firmicutes are fat-loving Gram-positive bacteria [88] this result suggests an inter-relationship of these bacteria and genetic selection for fast-growing characteristics of these broilers by the industry. In a recent comparative study [13], changes in the proportion of intestinal lactobacilli, as well as the total number of operational taxonomic units (OTU) between the three commercial broiler breeds were observed. Candidatus Arthromitus is a group of non-cultivable, spore-forming, Clostridium-related, commensal segmented filamentous bacteria (SFBs) that colonizes in the digestive tracts of animal species, and has been identified in three commercial broiler breeds [89]. As the core OTU, C. Arthromitus has been identified in all three groups of the Cobb and Hubbard broilers [13]. The most intriguing feature of SFBs is their close interaction with epithelial cells in the terminal ileum and their intimate cross talk with the host immune system. C. Arthromitus belongs to gut-indigenous Clostridium that induce immune regulatory T (Treg) cells. Intestinal Treg cells express T cell receptors that recognize antigen derived from gut microbiota [90]. SFBs send signals to control the balance between IL-17-producing T helper (Th17) cells that sustain mucosal immunity, and forkhead box p3 in the intestine [90]. Our previous studies have also reported that chicken IL-17A transcripts increase in the duodenum and jejunum of E. maxima-infected chickens [13, 91] where early inflammatory response plays an important role for development of protection against Eimeria infection. Upon feeding a mixture of oleoresins from Capsicum/C. longa, there is a different shift in the bacterial community in all broiler breeds with NE. Therefore, co-infection with E. maxima and C. perfringens may influence the presence of C. Arthromitus and the host immune system in Ross chickens. It will be important to conduct further studies to investigate the functional immune modulatory effects of dietary phytonutrients on C. Arthromitus in genetically different broiler breeds. In conclusion, dietary phytonutrients exert beneficial effects on gut health to reduce the negative consequences of NE, and nutratherapeutics mechanism may involve altering gut microbial communities. Further studies on the effects of dietary phytonutrients on gut microbiota in commercial broiler breeds are needed to develop alternative ways to reduce or replace antibiotics in poultry disease control. Future studies on the role of the avian intestinal microbiome in immune regulation and host–pathogen interactions are expected to shed new light on the host response to NE that will be beneficial for practical poultry husbandry.