Phytochemicals as antibiotic alternatives to promote growth and enhance host health Hyun Lillehoj, Yanhong Liu, Sergio Calsamiglia, Mariano E. Fernandez-Miyakawa, Fang Chi, Ron L. Cravens, Sungtaek Oh, Cyril G. Gay

To cite this version:

Hyun Lillehoj, Yanhong Liu, Sergio Calsamiglia, Mariano E. Fernandez-Miyakawa, Fang Chi, et al.. Phytochemicals as antibiotic alternatives to promote growth and enhance host health. Veterinary Research, BioMed Central, 2018, 49 (1), pp.76. ￿10.1186/s13567-018-0562-6￿. ￿hal-02973514￿

HAL Id: hal-02973514 https://hal.archives-ouvertes.fr/hal-02973514 Submitted on 21 Oct 2020

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REVIEW Open Access Phytochemicals as antibiotic alternatives to promote growth and enhance host health Hyun Lillehoj1*, Yanhong Liu2, Sergio Calsamiglia3, Mariano E. Fernandez‑Miyakawa4, Fang Chi5, Ron L. Cravens5, Sungtaek Oh1 and Cyril G. Gay6

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 scientifc developments on strategic antibiotic-free management plans, to evaluate regional diferences in policies regarding the reduction of antibiotics in animal agriculture and to develop antibiotic alterna‑ tives 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 diferent 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 strat‑ egies to facilitate the development of alternatives to antibiotic growth promoters (AGPs) without hampering animal production. The 3-day meeting consisted of four scientifc sessions including vaccines, microbial products, phyto‑ chemicals, 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 representa‑ tives 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.

Table of Contents 3.3 Use of phytonutrients in ruminants 1 Introduction 3.3.1 Essential oils as modifers of rumen 2 Plant‑derived phytochemicals as antibiotic alternatives fermentation 3 Examples of phytochemical antibiotic alternatives 3.3.2 Essential oils as modifers of metabolic in poultry and livestock production activities 3.1 Dietary phytochemicals enhancing innate immu‑ 4 Phytochemicals and the digestive microbiota nity in poultry 5 Examples of commercial phytochemicals and their 3.2 Dietary phytochemicals on weaning pig health synergistic action with other feed additives 5.1 Tannins in animal husbandry

*Correspondence: [email protected] 1 Animal Biosciences and Biotechnology Laboratory, Agricultural Research Service, US Department of Agriculture, Beltsville, MD 20705, USA Full list of author information is available at the end of the article

© The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/ publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lillehoj et al. Vet Res (2018) 49:76 Page 2 of 18

5.2 Synergistic action of phytochemicals with other mechanism of action, efcacy, and advantages and disad‑ feed additive antibiotic alternatives for commer‑ vantages of their applications in the feld. Furthermore, cial products the general consensus is that these products lack con‑ sistency and their efcacies vary among farms and loca‑ 5.3 Antibiotic alternatives: industry perspective tions. Terefore, their modes of action need to be better 6 Conclusions and future directions defned. Optimal combinations of various alternatives References coupled with good management and husbandry prac‑ tices will be the key to maximize performance and main‑ 1 Introduction tain animal productivity while we move forward, with Antibiotics, since their discovery in the 1920s, have the ultimate goal of reducing antibiotic use in the animal played a critical role in contributing to the economic industry. efectiveness of animal production as feed supplements at With declining AGPs usage and increasing consumers’ sub-therapeutic doses, to improve growth and feed con‑ concerns about superbugs, the quest for novel alternate version efciency, and to prevent infections [1]. In-feed replacements to mitigate antibiotic use in animal agricul‑ antibiotics (IFAs) are a common and well-established ture will grow signifcantly in the coming years. In this practice in the animal industry that has contributed to Phytochemical Session, we reviewed scientifc evidence the intensifcation of modern day livestock production. that phytochemicals stimulate innate immune cells, However, with intensifcation of animal agriculture, con‑ reduce oxidative stress, maintain gut integrity, promote cerns exist that the use of IFAs leads to development of benefcial bacteria growth, and reduce the negative con‑ antimicrobial resistance, posing a potential threat to sequences of infammation caused by enteric infections human health [2]. Although mixed opinions still exist as efective antibiotic alternatives to promote animal on the transfer of antibiotic resistance genes from ani‑ growth performance in poultry, swine, and beef and dairy mal pathogens to those of humans, studies have shown production. a potential link between the practice of using sub-thera‑ peutic doses of antibiotics and the development of anti‑ 2 Plant‑derived phytochemicals as antibiotic microbial resistance among the microbiota. alternatives In the US, antibiotic use in livestock and poultry feeds Phytochemicals, also referred to as phytobiotics or phy‑ is under scrutiny as a result of increasing consumer togenics, are natural bioactive compounds that are awareness and the demand for livestock products from derived from plants and incorporated into animal feed antibiotic-free production systems. In 2013, the US to enhance productivity [2]. Ideal antibiotic alternatives Food and Drug Administration (FDA) called for major should have the same benefcial efects of AGPs, ensure manufacturers of medically important animal drugs to optimum animal performance, and increase nutrient voluntarily stop labeling them for animal growth pro‑ availability. Considering the proposed mechanism of motion [3], and published its fnal rule of the Veterinary action of AGPs in modulating the gut microbiome and Feed Directive (VFD) in 2015. Te quest for alternative immunity, a practical alternative should exert a positive products has clearly intensifed in recent years with the impact on feed conversion and/or growth [2, 4]. Phyto‑ increase in regulations regarding the use of antibiotic chemicals can be used in solid, dried and ground form or growth promoters (AGPs) and the rise in consumer as extracts (crude or concentrated), and also can be clas‑ demand for poultry products from “Raised Without sifed as essential oils (EOs; volatile lipophilic substances Antibiotics” or “No Antibiotics Ever” focks [2]. obtained by cold extraction or steam/alcohol distillation) Tere has been a signifcant increase in scientifc and oleoresins (extracts derived by non-aqueous sol‑ papers in the recent literature on antibiotic alternatives vents) depending on the process used to derive the active and feed additives to promote growth and enhance gut ingredients [2]. Te main bioactive compounds of the health, and reduce the use of antibiotics in animal pro‑ phytochemicals are polyphenols, and their composition duction. Te classes of antibiotic alternatives that are and concentration vary according to the plant, parts of available to increase animal productivity and help poultry the plant, geographical origin, harvesting season, envi‑ and pigs perform to their genetic potential under exist‑ ronmental factors, storage conditions, and processing ing commercial conditions include probiotics, organic techniques [2]. acids, phytogenics, prebiotics, synbiotics, enzymes, anti‑ In recent years, phytochemicals have been used as nat‑ microbial peptides, hyperimmune egg antibodies, bac‑ ural growth promoters in the ruminants, swine and poul‑ teriophages, clay and metals [2]. Although the benefcial try industries. A wide variety of herbs and spices (e.g., efects of many of the alternatives developed have been thyme, oregano, rosemary, marjoram, yarrow, garlic, well demonstrated, there is a lack of information on their ginger, green tea, black cumin, coriander and cinnamon) Lillehoj et al. Vet Res (2018) 49:76 Page 3 of 18

have been used in poultry for their potential application addition, phytochemicals in Allium hookeri improved gut as AGP alternatives [2]. In contrast, several other phyto‑ barrier function, as demonstrated by increased expres‑ chemicals such as grape pomace, cranberry fruit extract, sion of gut tight junction proteins in the mucosa of Macleaya cordata extract, garlic powder, grape seed lipopolysaccharide (LPS)-treated young broiler chickens extract, and yucca extract, when tested as growth pro‑ [18]. moters, did not show any efects on performance param‑ eters [2]. In addition to herbs and spices, various EOs 3 Examples of phytochemical antibiotic (thymol, carvacrol, cinnamaldehyde, and eugenol, cori‑ alternatives in poultry and livestock production ander, star anise, ginger, garlic, rosemary, turmeric, basil, 3.1 Dietary phytochemicals enhancing innate immunity caraway, lemon and sage) have been used individually or in poultry as blends to improve animal health and performance [2]. A growing body of scientifc evidence has demonstrated Variable results have been reported with the use of EOs that many of the health-promoting activities of phyto‑ in poultry diets, some including cinnamaldehyde [5–7], chemicals are mediated through their ability to enhance and a blend of thymol and cinnamaldehyde improved host defense against microbial infections [4, 19]. Te body weight gain in broilers, while others like thymol immune-activating properties of medicinal plants such and EOs from star anise improved feed efciency, as seen as dandelion (Taraxacum ofcinale), mustard (Brassica by reduced feed conversion ratio (FCR). Curcuma alone juncea) and safower (Carthamus tinctorius) have been or curcuma with capsicum [7, 8] enhanced resistance to evaluated in vitro using avian lymphocytes and mac‑ enteric diseases such as coccidiosis and necrotic enteri‑ rophages [9]. All three extracts inhibit tumor cell growth, tis. Te variation in the results could be attributed to dif‑ stimulate innate immunity and exert antioxidant efects ferences in the composition, type and origin of the EOs in poultry [9]. Benefcial efects of cinnamaldehyde ((2E)- that were used, inclusion level, and the environmental 3-phenylprop-2-enal), a constituent of cinnamon (Cin- conditions of the trials [2]. Nevertheless, one commercial namomum cassia), a widely used favoring compound blend of phytonutrients (containing carvacrol, cinnamal‑ that has been traditionally used to treat human diseases, dehyde and Capsicum oleoresin), which enhances innate has been investigated. Cinnamaldehyde stimulated pri‑ immunity and reduces negative efects of enteric patho‑ mary chicken spleen lymphocyte proliferation in vitro gens [9, 10], was approved in the EU as the frst botanical and activated macrophages to produce high nitric oxide feed additive for improving performance in broilers and (NO) [6, 9]. livestock. Several trials performed with this commercial Because of increased regulation of AGPs in poultry blend have demonstrated consistent improvement in production, control of enteric diseases such as necrotic growth and feed efciency [9–11]. A meta-analysis of 13 enteritis (NE) and coccidiosis, which have been tradition‑ broiler studies involving the use of this commercial blend ally controlled by in-feed antibiotics [2], needs antibiotic- showed that its inclusion in diets increased body weight free disease control strategies. Although plant-derived gain and decreased feed conversion ratio and mortality chemicals with potent medicinal properties are currently [12]. in clinical trials for treatment of a variety of diseases in Te mechanism of action of phytochemicals is not humans, only limited research has documented the ben‑ clearly understood but may depend upon the composi‑ efcial efects of phytochemicals on avian diseases [4, tion of the active ingredients in the product being used. 19]. Dietary supplementation of 1-day-old chickens with Te benefcial efects of phytochemicals are attributed to cinnamaldehyde at 14.4 mg/kg showed up to 47-fold their antimicrobial and antioxidant properties. In addi‑ greater levels of gene transcripts encoding interleukin tion, the inclusion of phytochemicals in the diets alters (IL)-1β, IL-6, IL-15 and interferon (IFN)-γ in intestinal and stabilizes the intestinal microbiota and reduces lymphocytes, compared with chickens given a standard microbial toxic metabolites in the gut, owing to their diet [15, 19]. Cinnamaldehyde-fed chickens showed 17 direct antimicrobial properties on various pathogenic and 42% increased body weight gains following Eimeria bacteria, which results in relief from intestinal challenge acervulina and E. maxima infections, respectively, 40% and immune stress, thus improving performance [13]. reduced E. acervulina oocyst shedding, and 2.2-fold Another important benefcial efect of dietary inclusion higher E. tenella-stimulated parasite antibody responses, of phytochemicals is reduction in oxidative stress and compared with the control. Te most reliable genetic net‑ increase in antioxidant activity in various tissues, and work induced by dietary cinnamaldehyde treatment is thus, improved health [14]. Phytochemicals also exert related to antigen presentation, humoral immunity, and their action through immunomodulatory efects such as infammatory disease. Chickens continuously fed 15 mg/ increased proliferation of immune cells, modulation of kg anethole from hatch and orally challenged with live E. cytokines, and increased antibody titers [5–8, 15–18]. In acervulina oocysts showed increased body weight gain, Lillehoj et al. Vet Res (2018) 49:76 Page 4 of 18

decreased fecal oocyst excretion, and greater anti-para‑ oleoresin-supplemented diet had increased numbers site serum antibody responses, compared with the con‑ of intestinal T cells, compared with untreated controls. trol group. Global gene expression analysis by microarray While numerous studies have shown disease preven‑ hybridization in the intestinal lymphocytes of anethole- tion or immune-enhancing efects of phytochemicals, fed birds showed that many genes related to the infam‑ few have examined the underlying mechanisms that are matory response are altered [17]. Te levels of transcripts involved. Some phytochemicals inhibit innate immune encoding IL-6, IL-8, IL-10 and TNF superfamily member response by targeting pathogen pattern recognition 15 (TNFSF15) in intestinal lymphocytes were increased receptors or their downstream signaling molecules [20]. in parasite-infected chickens given the anethole-con‑ Te Clostridium-related poultry disease such as NE taining diet, compared with the control chickens given a causes substantial economic losses on a global scale [21]. standard diet. It has been suggested that dietary phytonutrients could Garlic metabolites also have been tested in poultry be used against NE. Supplementation of a mixture of using propyl thiosulfnate (PTS) and propyl thiosulf‑ Capsicum and Curcuma longa oleoresins (XTRACT®​) nate oxide (PTSO) [16]. Supplementation of 10 mg/kg from hatch increased body weight and reduced gut lesion PTS/PTSO increased body weight gain and serum anti‑ scores in NE-aficted birds, compared with infected birds body titers against proflin, an immunogenic protein of given the non-supplemented diet [7]. Te XTRACT®​ -fed Eimeria, and decreased fecal oocyst excretion in E. acer- birds also had lower serum α-toxin levels and reduced vulina-challenged chickens compared with chickens fed mRNA expression of IL-8, lipopolysaccharide-induced a control diet [16]. Te addition of PTS/PTSO in broil‑ TNF factor (LITAF), IL-17A and IL-17F in intestine, er’s diet altered many genes related to innate immunity, but increased cytokine/chemokine levels in splenocytes, including TLR3, TLR5 and NF-κB [16] and down-regu‑ compared with birds fed with the control diet. Tis lated expression of IL-10 compared with the control diet. study documented the molecular and cellular immunity In uninfected chickens, dietary supplementation with changes following dietary supplementation with extracts PTS/PTSO increased the levels of transcripts encoding of Capsicum and turmeric that may be relevant to pro‑ IFN-γ, IL-4, and an antioxidant enzyme, paraoxonase 2, tective immunity against avian NE [7]. Future studies but decreased transcripts for peroxiredoxin-6 [16]. are needed to defne the molecular and cellular mode of Combination of multiple phytochemicals exert syner‑ action of this phytochemical combination for the control gistic efects to reduce negative consequences of enteric of NE in the feld. infections. Dietary supplementation of newly hatched broiler chickens with a mixture of Curcuma longa, Cap- 3.2 Dietary phytochemicals on weaning pig health sicum annuum (pepper), and Lentinus edodes improved Phytochemicals have been used for human nutrition and body weight gain and serum antibody titers against pro‑ health improvement due to their potential biological flin, and reduced fecal oocyst shedding in E. acervulina- functions, such as, antiviral, antimicrobial, antioxidant infected birds, compared with the birds fed the control and anti-infammatory efects [2, 5, 22]. Various phyto‑ diet or a diet containing Capsicum plus Lentinus [5]. chemicals exhibit a wide spectrum of antibacterial activi‑ Te efects of carvacrol, cinnamaldehyde and Capsicum ties against Gram-negative and Gram-positive bacteria oleoresin on the regulation of expression of genes asso‑ [23] with several diferent modes of action. First, phyto‑ ciated with immunology, physiology, and metabolism chemicals directly kill bacteria due to their hydropho‑ have been investigated in chickens using high-through‑ bicity, which enables them to partition into the lipids of put microarray analysis [15]. Te levels of transcripts the bacterial cell membrane and mitochondria, resulting for IL-1β, IL-6, IL-15 and IFN-γ in gut lymphocytes in leakage of critical intracellular materials [24]. Second, were also greater in the Curcuma/Capsicum/Lentinus- phytochemicals contain a high percentage of phenolic fed birds, compared with those fed the standard, Cur- compounds, which possess strong antibacterial proper‑ cuma or Capsicum/Lentinus diet. In a follow-up study, ties [25]. Tird, the active components in phytochemicals a combination of carvacrol, cinnamaldehyde and Cap- disturb the enzyme system of bacteria and block their sicum oleoresin, or a mixture of Capsicum and Cur- virulence [26]. Fourth, certain bioactive components in cuma oleoresins increased protective immunity against phytochemicals may prevent the development of viru‑ experimental E. tenella infection following immuni‑ lence structures in bacteria, such as fagella, which criti‑ zation with proflin, compared with untreated and cal for bacterial adhesion [27]. immunized controls [10]. Immunized chickens fed the Phytochemicals are also proposed for use as antioxi‑ carvacrol/cinnamaldehyde/Capsicum-supplemented dants in animal feed, which will protect animals from diet showed increased numbers of macrophages in the oxidative damage caused by free radicals. Te antioxi‑ intestine, while those given the Capsicum/Curcuma dative properties of extracts of oregano, thyme, clove, Lillehoj et al. Vet Res (2018) 49:76 Page 5 of 18

pepper, lavender and basil have been evaluated by many decreases ileal total microbial mass and increases the studies in vitro [28, 29]. Our recent in vitro assays have lactobacilli:enterobacteria ratio. Michiels et al. [38] have also revealed that EOs extracted from peppermint also indicated that supplementing with 500 ppm car‑ and spearmint have cellular antioxidant activities by vacrol and thymol reduces the number of intraepithelial increasing intracellular glutathione concentration in lymphocytes and increases villus height/crypt depth in ­H2O2-stimulated intestinal epithelial cells (unpublished the distal small intestine. data). Frankič et al. [30] showed that supplementation of Escherichia coli post-weaning diarrhea is a common phytochemicals to pigs reduced DNA damage in lympho‑ cause of death in weaned pigs. Tis diarrhea is respon‑ cytes, which indicates their potentially benefcial efects sible for economic losses due to mortality, morbidity, on the immune system under dietary-induced oxida‑ decreased growth performance, and cost of medication tive stress. Te antioxidant activity of phytochemicals is [39]. Enterotoxigenic E. coli are the most dominant types highly correlated with their chemical composition [31]. of pathogenic E. coli that cause diarrhea in both pre- and Phenolic OH groups in thymol, carvacrol and other phy‑ post-weaning piglets [40]. Capsicum oleoresin, garlicon, tochemicals act as hydrogen donors to the peroxy radi‑ and turmeric oleoresin have been tested in an in vivo cals produced during the frst step in lipid oxidation, thus pathogenic E. coli challenge study to determine the efects retarding ­H2O2 formation [32]. of individual phytochemicals on diarrhea and gut health Te anti-infammatory efects of phytochemicals have of weaning pigs [41]. Te pigs were weaned at 21 days of been widely reported in in vitro cell culture models. age, transported to the experimental facility, and given EOs from clove, tea, garlic, cinnamon and others have the experimental diets immediately. After a 5-day adap‑ potential anti-infammatory activities and suppress the tation period, they were challenged with three consecu‑ production of TNF-α, IL-1β and NO from LPS-induced tive daily doses of 10­ 10 colony forming units/3 mL of a mouse macrophages [33]. Our previous research in vitro hemolytic E. coli with F18 fmbria. Te experimental with porcine alveolar macrophages showed that carvac‑ diets were a control diet based on corn and soybean meal rol, Capsicum oleoresin, cinnamaldehyde, garlic, eugenol, and three additional diets containing 10 mg/kg of each anethol, and turmeric oleoresin suppress the produc‑ plant extract. Te E. coli infection increased diarrhea tion of proinfammatory cytokines (TNF-α and IL-1β) score, frequency of diarrhea, and reduced growth rate, from LPS-stimulated macrophages [22], which indicates feed efciency and villus height of the small intestine. that all of these phytochemicals have anti-infammatory However, supplementation with individual phytochemi‑ efects. Te modes of action for the anti-infammatory cals reduced overall frequency of diarrhea of pigs, indi‑ activities of phytochemicals are not clear, but evidence cating that feeding phytochemicals may enhance disease suggests that these efects are partially mediated by resistance in pigs. Supplementation with phytochemicals blocking the nuclear factor (NF)-κB activation pathway also improved ileal villus height and upregulated mRNA [34]. For example, curcumin can block cytokine-induced expression of the MUC-2 gene, which indicated that NF-κB DNA binding activity, RelA nuclear translocation, the reduced diarrhea score was likely due to improved IκBα degradation, IκB serine 32 phosphorylation, and gut barrier function and integrity. Pigs infected with E. IκB kinase activity [34]. coli showed an increased number of white blood cells, Weaning is one of the most challenging and critical serum proinfammatory cytokine (TNF-α) and acute stages in swine production. Its efects are multifacto‑ phase protein (haptoglobin) and increased recruitment of rial, including behavior, environment, disease, immunity macrophages and neutrophils in the ileum. Dietary sup‑ and nutrition. In this period, piglets are immediately plementation with phytochemicals reduced white blood subjected to a combination of stressors that predispose cells, neutrophils, serum TNF-α and haptoglobin and the them to diarrhea, which can adversely afect survival at numbers of macrophages and neutrophils in the ileum an early and most vulnerable stage [35]. Te benefcial compared with the control diet. Tese observations indi‑ efects of phytochemicals on weaning pigs have been cate that feeding low doses of phytochemicals reduces reported by diferent research groups. Manzanilla et al. both systemic and local infammation caused by E. coli [36] and Nofrarías et al. [37] have suggested that phyto‑ infection. chemicals improve gut health. Tey have reported that To decipher the underlying mechanism behind the a mixture of phytochemicals (XTRACT​®) standard‑ benefts of feeding phytochemicals, microarray analysis ized to 5% (w/w) carvacrol, 3% cinnamaldehyde, and has been conducted to characterize gene expression in 2% Capsicum oleoresin (oregano, cinnamon and Mexi‑ the ileal mucosa of pigs experimentally infected with E. can pepper) increases stomach contents, suggesting an coli. Microarray results indicate that feeding phytochem‑ increased gastric retention time. In addition, XTRACT®​ icals enhances the integrity of membranes, especially Lillehoj et al. Vet Res (2018) 49:76 Page 6 of 18

several tight junction proteins. Supplementation of phy‑ processes in ruminants is still the most efcient strategy tochemicals downregulates expression of genes related to to improve animal performance. antigen processing and presentation and other immune- AGPs are efcient in shifting rumen fermentation response-related pathways, indicating that these phyto‑ towards more efcient energy and nitrogen utilization chemicals attenuate the immune responses caused by E. pathways [47], improving productivity in dairy and beef coli infection [42]. diets [48, 49]. Terefore, industry is searching for alter‑ Another in vivo study on porcine reproductive and native feeding strategies and/or additives that will allow respiratory syndrome virus (PRRSV) [43] showed that it to maintain the current level of production without feeding Capsicum oleoresin, garlicon, and turmeric ole‑ increasing the cost. oresin to weaning pigs enhances the immune responses Phytonutrients are a group of small organic mole‑ to PRRSV challenge and may help alleviate the nega‑ cules present in plants that modify the nutritional value tive impact of infection, as indicated by reduced viral of feeds by either modulating the digestion of nutrients load and serum concentrations of infammatory media‑ in the digestive tract, or other systemic metabolic path‑ tors, and shortened duration of fever. In summary, phy‑ ways. Some phytonutrients have a strong antimicrobial tochemicals are strong candidates to replace antibiotics activity [50]. However, these molecules are not suitable to improve growth performance and health of pigs. Te for use in ruminants because the activity of rumen bac‑ potential benefts of plant extracts may difer due to the teria is essential for the proper function of the rumen. large variation in the composition of plant extracts. Tis Research on alternatives to antibiotics as feed supple‑ diversity prompts us to select optimal feed additives for ments in cattle should focus on molecules and doses evaluating their possible roles as alternatives to antibiot‑ that are able to produce subtle changes in the microbial ics in swine production. metabolism and modify their rate of growth [51]. In the context of the continuous fow in the rumen, a change 3.3 Use of phytonutrients in ruminants in growth rates results in changes in the proportion In ruminants, the host and rumen microorganisms of rumen bacteria populations, resulting in changes establish a symbiotic relationship by which the animal in the fermentation profle. For example, Patra and Yu provides nutrients and the proper fermentation condi‑ [52] were able to prove how diferent phytonutrients tions, and microbes degrade fber and synthesize micro‑ have diferent capacities in modifying the structure of bial protein as an energy and protein supply for the host, the microbial population of the rumen. Tese changes respectively. Carbohydrates are fermented in the rumen are large in oregano (where thymol and carvacrol are into pyruvate, resulting in the production of metabolic the main active components) and peppermint (where hydrogen. Volatile fatty acids (VFAs) are natural hydro‑ menthol and menthone are the main active compo‑ gen sinks that help maintain the equilibrium of hydro‑ nents) oils, but smaller, and more adequate, in clove gen and the fermentation process active. Retention of bud (where eugenol is the major active component) energy from glucose is the highest in propionate (109%), and garlic oils. Ferme et al. [53] also have demonstrated intermediate in butyrate (78%) and the lowest in ace‑ that the reduction in protein degradation and ammo‑ tate (62.5%). Although methane is efective in retaining nia production is achieved through changes in the total hydrogen, the energy retained is lost through eructation amount of Prevotella ssp. in the rumen; a major group and not available to the host. Manipulation of the rela‑ of bacteria involved in amino acid deamination. Tese tive proportions of these VFAs is key to the development fndings are important to set clear objectives in the of targets to modify rumen microbial fermentation [44]. search for alternatives to AGPs, which should identify Protein degradation is also important for the supply of phytonutrients that can modify the VFA proportions nitrogen to rumen microbes for their growth, but excess and protein degradation in the rumen without afecting ammonia nitrogen is absorbed through the rumen wall, nutrient degradation and the normal function of the transformed into urea in the liver, and excreted through rumen. the urine. In most production systems, ammonia nitro‑ Most phytonutrients of interest in animal feeding gen in the rumen is produced in excess of the ability of are classifed into three main groups: saponins, tannins rumen microbes to use it, resulting in signifcant produc‑ and EOs. Saponins and sarsaponins are the main active tion costs and an increase in the release of nitrogen into components of several phytochemicals, including yucca, the environment [45]. Terefore, controlling proteolysis, quillaja, alfalfa and fenugreek. Saponins exhibit antibac‑ petidelysis and deamination should also be considered terial [54] and antiprotozoal [54, 55] activity, resulting targets of interest in the modulation of rumen fermenta‑ in a reduction in ammonia nitrogen concentration. Tan‑ tion [44]. In fact, in a recent study, Van der Aar et al. [46] nins are phenolic compounds found in almost every plant indicated that improving the efciency of the digestion part, and are divided into two groups, hydrolysable and Lillehoj et al. Vet Res (2018) 49:76 Page 7 of 18

condensed tannins. Condensed tannins have the abil‑ of large peptides to amino acids and small peptides [59]. ity to bind and precipitate proteins and may be useful in Te combination of eugenol and cinnamaldehyde may the control of protein utilization by ruminants [56], but work in synergy to inhibit peptidolysis and deamination, at high levels may interfere with dry matter (DM) intake and then improve the overall supply of amino acids and and digestibility of nutrients [56], and may decrease small peptides to microorganisms and the host. Tere‑ the incidence of bloating [55]. EOs are secondary plant fore, a synergetic advantage could be expected by com‑ metabolites present in many plants and may have a wide bining specifc phytonutrients that work at diferent range of efects. In this section, we review recent research levels in the same metabolic pathway. on the use of EOs as feed additives in ruminants. Tere are limited data reported about the efects of phytochemicals on performance of ruminants. Feeding 3.3.1 Essential oils as modifers of rumen fermentation cinnamaldehyde alone or in combination with eugenol Te increased rumen fermentation is indicated by the results in increased in milk production of 1.7–2.7% [64]. increase in propionate and decrease in methane, acetate An even better response is reported when a combination and ammonia nitrogen, without reducing total VFA [57] of cinnamaldehyde, eugenol and capsicum is fed to dairy in the in vitro fermentation system. When phytochemi‑ cattle, with increases in energy-corrected milk produc‑ cals are tested, a considerable variation in fermentation tion of 5.2% [65] and 3.2% [66]. However, no diferences with diferent extracts is observed due to the content of have been observed in most of cases due to the small size active compounds in these extracts [58]. Terefore, it of the studies. Bravo et al. [67] have summarized a large is necessary to either report the concentration of these set of in vivo feld trials using combinations of cinnama‑ active compounds in phytochemicals, or use the active dehyde and eugenol through a meta-analysis, and have components to defne activities, doses and mechanisms reported an improvement in milk production of 3.0% for of action in an unequivocal form. dairy cattle. For example, garlic oil reduces the proportions of ace‑ tate and branched-chain VFAs, and increases the propor‑ 3.3.2 Essential oils as modifers of metabolic activities tions of propionate and butyrate in vitro [57, 59], and the Many phytonutrients have metabolic efects that are not fermentation profle is consistent with changes observed related to their activities in the rumen [68, 69]. Prelimi‑ when methane inhibitors are supplied to ruminants. nary in vitro rumen fermentation studies in dairy cattle Te anti-methanogenic efect of garlic and its active have not identifed capsicum as a potential modifer for components is the result of direct inhibition of Archea rumen function [61, 70]. Capsicum increases DM and microorganisms in the rumen through the inhibition of water intake in beef cattle from 9.2 to 14% [70–72], while hydroxymethylglutaryl coenzyme A (HMG-CoA) reduc‑ these efects are not observed in dairy cattle [73, 74]. Te tase; a specifc pathway essential for the membrane sta‑ benefts may be more signifcant when intake is compro‑ bility of Archea [57, 59]. Tis observation was supported mised, such as when the cattle arrive at feedlots or during by Miller and Wolin [60], who reported similar efects heat stress. Te increase in DM intake patterns is prob‑ when using statins, known to inhibit HMG-CoA reduc‑ ably also related to a more stable rumen pH [75]. tase. However, benefts are often inconsistent, and strong Capsicum has been reported to modulate immune inhibition of VFA production by garlic oil has been function [42]. Oh et al. [76] have reported an improve‑ reported in some cases [59, 61, 62]. Te variable efects ment in immunity indicators, with an increase in neu‑ of garlic oil on total VFA production is likely due to the trophils and decrease in lymphocytes when cattle are fed short margin of safety in the doses between adequate and rumen-protected capsicum. Feeding rumen-protected toxic levels. capsicum is reported to improve milk production. Stel‑ Cinnamaldehyde and eugenol also reduce the molar wagen et al. [77] and Wall et al. [78] have reported proportion of acetate, and increase the molar proportions increases in milk production of 6.6 and 9.1% in pasture of propionate and butyrate [59, 61]. Tese observations and intensive production systems, respectively. Another are consistent with improved energy retention by those three studies have also reported that supplementation phytochemicals and potentially due to the inhibition of rumen-protected capsicum improved milk produc‑ of methanogenesis [63]. Cinnamaldehyde also reduces tion by 6.2% [76], 10% [79], and 4.4% [80], respectively. ammonia nitrogen and increases free amino acids, sug‑ Te average increase in milk production in those studies gesting that deamination of amino acids is inhibited was higher than the efects attributed to the modulation in the rumen [59, 61]. Ferme et al. [53] have reported of rumen fermentation. Oh et al. [80] observed that sup‑ that cinnamaldehyde reduces Prevotella spp., bacteria plementation with rumen-protected capsicum resulted involved in deamination, in an in vitro rumen simula‑ in a lower insulin concentration after a glucose toler‑ tion system. However, Eugenol inhibits the breakdown ance test. Tese results suggest that capsicum modifes Lillehoj et al. Vet Res (2018) 49:76 Page 8 of 18

glucose metabolism, redirecting glucose away from Te intestinal microbiota plays a critical role in infam‑ peripheral tissues and towards the mammary gland to matory gut diseases of humans and animals [86]. Recent increase milk production. In fact, Bovine somatotropin development and application of next-generation sequenc‑ (bST) increases milk production by an average of 13%, ing technologies using 16S rRNA gene have allowed redirecting glucose to the mammary gland, although the investigation of the signifcant roles of the microbiota in mechanism of action is diferent. Tis is an exciting new gastrointestinal tract diseases, and have facilitated inves‑ application of phytonutrients that presents an oppor‑ tigation of host–pathogen interaction in NE [86]. Te tunity to improve production, not only by reducing the efect of dietary phytochemicals on gut microbiota was use of antibiotics, but also by providing an alternative to studied in three major commercial broiler chickens fed the use of some hormones. Te average efect of rumen with Capsicum and C. longa oleoresins [13]. Among the modifers like monensin, yeast or some phytonutrients, three chicken breeds, Cobb, Hubbard and Ross, oleoresin commonly increase milk production by 2–4%, while cap‑ supplementation was associated with altered intestinal sicum increases milk production by an average of 7%. microbiota. Te results suggested that dietary feeding of Capsicum and C. longa oleoresins reduces the negative 4 Phytochemicals and the digestive microbiota consequences of NE, in part, through alteration of the Te mammalian gastrointestinal tract harbors a dense gut microbiome. Although these are preliminary charac‑ and diverse microbial community, which is composed terizations of the efects of dietary phytochemicals on gut primarily of bacteria but also includes fungi, Archaea microbiota but document the role of dietary Capsicum and viruses. Collectively, these are referred to as intesti‑ and C. longa oleoresins in regulating disease susceptibil‑ nal microbiota. Tese microorganisms are environmen‑ ity to NE via altering the intestinal microbiota in com‑ tally acquired, and their metabolic functions can shape mercial broiler chickens. host physiology. Many vertebrates consume a diet rich A recent study [13] showed that Firmicutes was the in complex nutrients that are indigestible by their own dominant phylum and Lactobacillus was the predomi‑ intestinal enzymes, relying on the diverse biochemical nant genus identifed in the ileum in all broiler breeds catabolic activities of the microbiota. Available evidence and all treatment groups. Tese results are consistent strongly suggests that the gut microbiota plays important with previous studies that showed Lactobacillus as the roles in host energy harvest, storage and expenditure, as principal microorganism in the gastrointestinal tract of well as overall nutritional status [81–84]. It must be high‑ uninfected conventional broilers [87]. Because Firmicutes lighted that germ-free animals that lack any microbiota are fat-loving Gram-positive bacteria [88] this result sug‑ weigh less and have less fat than conventional animals gests an inter-relationship of these bacteria and genetic [85], pointing out a key role of the microbiota in weight selection for fast-growing characteristics of these broil‑ gain. Gut microbiota may afect weight gain through reg‑ ers by the industry. In a recent comparative study [13], ulating nutrient extraction, and modulating the immune changes in the proportion of intestinal lactobacilli, as system and metabolic signaling pathways [82]. well as the total number of operational taxonomic units Many classes of substances with antibiotic activity that (OTU) between the three commercial broiler breeds are efective for animal growth promotion display multi‑ were observed. Candidatus Arthromitus is a group of ple modes of action and spectra of activity over the gas‑ non-cultivable, spore-forming, Clostridium-related, com‑ trointestinal microbiota. It has been difcult to predict mensal segmented flamentous bacteria (SFBs) that col‑ which microbial changes are responsible for increases in onizes in the digestive tracts of animal species, and has weight gain, feed efciency or health promotion. Culture- been identifed in three commercial broiler breeds [89]. independent approaches using next-generation DNA As the core OTU, C. Arthromitus has been identifed in sequencing have provided researchers with a revolu‑ all three groups of the Cobb and Hubbard broilers [13]. tionary tool to look into microbiomes that could not be Te most intriguing feature of SFBs is their close inter‑ achieved before, and has begun to transform our view action with epithelial cells in the terminal ileum and of intestine-associated biodiversity of animal produc‑ their intimate cross talk with the host immune system. tion. Improving the understanding of microbiota and C. Arthromitus belongs to gut-indigenous Clostridium host metabolism would help to develop better strategies that induce immune regulatory T (Treg) cells. Intestinal and products for animal production and welfare, food Treg cells express T cell receptors that recognize antigen safety and public health. Te selection of microbes that derived from gut microbiota [90]. SFBs send signals to aid in nutrient extraction, regulating microbial carbohy‑ control the balance between IL-17-producing T helper drate, protein and lipid metabolism, and the prevention (T17) cells that sustain mucosal immunity, and forkhead of subclinical infections will help to promote productive box p3 in the intestine [90]. Our previous studies have parameters [83]. also reported that chicken IL-17A transcripts increase in Lillehoj et al. Vet Res (2018) 49:76 Page 9 of 18

the duodenum and jejunum of E. maxima-infected chick‑ (proteins and polysaccharides) and metal ions, which are ens [13, 91] where early infammatory response plays an commonly included in ruminant diets such as forage and important role for development of protection against sorghum. Tannins are chemically classifed as hydrolys‑ Eimeria infection. Upon feeding a mixture of oleoresins able or condensed based on their chemical structure, and from Capsicum/C. longa, there is a diferent shift in the are widely used to improve several aspects of animal hus‑ bacterial community in all broiler breeds with NE. Tere‑ bandry. Some tannins are potent antimicrobials, acting, fore, co-infection with E. maxima and C. perfringens may for example, by iron deprivation or interactions with vital infuence the presence of C. Arthromitus and the host proteins such as enzymes [95] or bacterial cell wall pro‑ immune system in Ross chickens. It will be important teins [96], displaying either bactericidal or bacteriostatic to conduct further studies to investigate the functional activities [97]. Gram-positive bacteria are particularly immune modulatory efects of dietary phytonutrients on sensitive to tannins [98]. C. Arthromitus in genetically diferent broiler breeds. In In ruminants, tannins modify the digestive processes conclusion, dietary phytonutrients exert benefcial efects not only by binding dietary protein (rumen bypass), on gut health to reduce the negative consequences of NE, but also through modulation of rumen microbiota and and nutratherapeutics mechanism may involve altering improvement of the growth of certain bacterial popula‑ gut microbial communities. Further studies on the efects tions [99]. Te efects of tannins on rumen microbiota of dietary phytonutrients on gut microbiota in com‑ may vary depending on the molecular nature of these mercial broiler breeds are needed to develop alternative polyphenols [99, 100]. Te understanding of in vivo inter‑ ways to reduce or replace antibiotics in poultry disease actions between rumen bacteria and sources of plant tan‑ control. Future studies on the role of the avian intestinal nins are limited. microbiome in immune regulation and host–pathogen Approximately 90% of total microbiota in the bovine interactions are expected to shed new light on the host rumen is composed by Firmicutes and Bacteroidetes, response to NE that will be benefcial for practical poul‑ with large inter-individual variance in their relative abun‑ try husbandry. dance, with a strong inverse correlation between abun‑ In foregut fermenters, such as cattle and sheep, up dance of both phyla [101]. In steers fed with a high-starch to 50% of their energy may be obtained from microbial diet, bacterial populations belonging to the Bacteroidetes metabolites [92], including VFAs. In contrast, hindgut were the most abundant in all animals (almost 50%) fermenters (such as pigs and chickens), in which most while Firmicutes accounted for ~40% of the total micro‑ fermentation takes place in the cecum and large intestine, biota. However, this predominance was inverted when a receive only 5–10% of energy demands from microbial blend of tannins were added to the feed, with a signif‑ fermentation products [93]. Although these diferences cantly higher percentage of Firmicutes and a reduction seem to be important from a functional point of view, in in Bacteroidetes. Accordingly, steers supplemented with ruminants or monogastrics, gastrointestinal microbiota tannins have a higher Firmicutes to Bacteroidetes (F/B) composition is similarly central to improved animal pro‑ ratio in comparison with the control group [101]. Many duction in both groups, and the impact of phytochemi‑ studies have reported that F/B ratio increases when body cals on these microbiota might be responsible for most of mass index is increased, and F/B ratio is higher in obese the positive efects observed. than in lean animals [102–104]. Te rational basis for the Many benefcial properties of plants are derived from apparent relation between F/B ratio and increase in body their specifc bioactive components, which are also weight is that Firmicutes are not as efective as Bacteroi- synthesized as chemical protectants against microbial detes at gathering energy from digesta for themselves, infection. Te most important useful phytochemicals leaving more energy to be absorbed by the host. with antimicrobial activities can be divided into several Diversity of rumen microbiota is one of the key features categories, such as phenolics/polyphenols, terpenoids/ in ruminant animals, which confers upon cattle the abil‑ essential oils, alkaloids, and lectins/polypeptides [94]. ity to adapt to a wide range of dietary conditions [105]. Some compounds among these categories are known to Dietary quebracho and chestnut tannins diminish rumen be important for improving animal production, as well richness but do not signifcantly afect the complexity of as inducing an extensive number of health-promoting the bacterial communities (i.e. balance between the rela‑ efects. Tannins and EOs are fed commercially to several tive abundances of bacterial taxa). Tere is an increase domestic animal species and, as growth promoters, they in rumen microbiota richness but no change in Shan‑ modify the gut microbiota in diferent ways. non’s diversity index after supplementation with a blend Tannins are a complex group of polyphenolic com‑ of polyphenols and EOs in dairy heifers fed a high-grain pounds found in many plants species, functionally diet, supporting the idea that polyphenols can modu‑ defned by their capacity to complex macromolecules late bacterial richness without disrupting the overall Lillehoj et al. Vet Res (2018) 49:76 Page 10 of 18

structure of the rumen microbiota population. Similarly, Treponema is also reduced by tannins. Among Veil‑ β-diversity analysis of rumen samples of steers fed with lonellaceae members, Succiniclasticum, which specializes chestnut and quebracho showed no signifcant changes in fermenting succinate to propionate, doubles its levels in bacterial diversity compared with the control group in tannin-treated animals. Lipolytic genus Anaerovi- [101]. Low microbial richness in the rumen is closely brio is signifcantly enhanced by tannins. Selenomonas is linked to a higher feed efciency in dairy cows [106]. Te also increased in tannin-supplemented animals. Among authors have suggested that lower richness in the rumen ureolytic bacteria, Butyrivibrio is the most abundant and of efcient animals results in a simpler metabolic net‑ it is negatively afected by tannin treatment, as well as work, which leads to higher concentrations of specifc Treponema and Succinivibrio. Methanogens belonging to metabolic components that are used to support the host’s the phylum Euryarchaeota are less abundant in tannin- energy requirements. Diversity analysis indicate that bac‑ supplemented steers and their levels are inversely corre‑ terial richness is lowered by tannins, but the overall bac‑ lated with rumen pH. Methanosphaera is also reduced by terial complexity of the rumen is not signifcantly afected tannins. Current literature indicates that tannins can be by chestnut and quebracho tannins supplementation. supplemented to improve the sustainability of both dairy Several studies have found an increase of rumen pH, and beef cattle by reducing methane emissions and nitro‑ decrease of ammonia concentration, and lower meth‑ gen excretion, and enhancing animal performance. ane emissions after feed supplementation with several In monogastrics, that is, broiler chickens, tannins tannins including chestnut and quebracho, resulting obtained from several sources seem to improve growth in a reduction of protein degradation and therefore performance and reduce the detrimental efects of path‑ an improvement in nitrogen utilization in the rumen ogenic bacterial species such as C. perfringens [101]. [107]. Tannins are considered as alternative agents to Te establishment of a stable microbiota is a complex antibiotics, they improve animal health and produc‑ process that is infuenced by various factors, including tive performance while suppressing methanogenesis. genetic lineage, age, diet, use of growth promoter anti‑ Tese observations could be explained by changes in biotics, probiotics, litter composition, stress and disease the microbiota in the rumen. Signifcant changes in the [86, 109–111]. Terefore, any alteration in the intestinal abundance of certain taxa have been detected in tannin- microbiota may have functional consequences to the treated steers. Among Bacteroidetes, Prevotella was the health of the host and, therefore, productivity. most abundant genus, accounting for >40% of this phy‑ Te broiler chicken gastrointestinal tract is colonized lum. Te abundance of Prevotella was lower in tannin- by a dense community of microorganisms that is inti‑ supplemented animals than in the control group. In mately connected to the global heath and development contrast, Clostridia was the predominant class, which of the host. Te cecum houses the highest microbial cell accounted for >90% of total Firmicutes, and it was sig‑ densities of the chicken gut and performs key process for nifcantly enhanced in tannin-treated animals. Among birds such as the fermentation of cellulose, starch and Clostridia, Ruminococcaceae was the most abundant other resistant polysaccharides [86]. A principal coordi‑ family and showed a signifcantly higher abundance in nate analysis (PCoA) based on unweighted UniFrac dis‑ tannin-supplemented animals. Within the Ruminococ‑ tances was conducted to determine any diferentiation caceae, most of the sequences obtained in untreated ani‑ between sample clusters of tannin-treated versus anti‑ mals belonged to unclassifed members and the genus biotic-growth-promoter-treated versus untreated birds. Ruminococcus, and both taxa were enhanced in tannin- PCoA plots revealed that the samples corresponding to treated steers. Other non-clostridial bacteria within the each dietary treatment shaped distinct series, suggesting phylum Firmicutes were signifcantly altered by tannins, that tannins diferentially modulate cecal microbiota. including members of class Erysipelotrichi. Members of High-throughput sequencing of 16S rRNA gene ampli‑ class Bacilli (Streptococcus and Lactobacillus) showed cons has been used to identify functional diversity [112] moderate increases in their abundance in tannin-treated or variability [113] of the microbiome in the gut of animals. Genus Fibrobacter was signifcantly afected by broiler chickens. In most studies related to tannins, cecal tannins, accounting for 0.10% of total microbiota in the microbiota in chickens was dominated by Firmicutes control animals and only 0.005% in tannin-treated ani‑ and Bacteroidetes [114, 115], comprising >80% of the mals. Other minor fbrolytic bacteria were more abun‑ microbiota. Te most abundant Bacteroidetes detected dant in tannin-treated steers, including the genus Blautia in cecal contents belonged to genus Bacteroides and an and member of the Eubacteriaceae genus Anaerofustis. unclassifed genus of the family Barnesiellaceae. Among Tannins remodel the bacterial ecosystem of the rumen, the Firmicutes, order Clostridiales and family Rumino‑ particularly the niche of fber and starch degradation, and coccaceae were the most abundant taxa. Te F/B ratio the methanogenic bacteria [108]. Lillehoj et al. Vet Res (2018) 49:76 Page 11 of 18

was signifcantly higher in tannin-fed animals than in the health and production. We are just barely starting to control or antibiotic growth promoter groups. understand the dynamics between the highly complex Bacteroides is a Gram-negative genus that utilizes connection between environment, host and microbi‑ plant glycans as its main energy sources. Bacteroides ota. More information is necessary to clarify how we is one of the main bacteria involved in producing can manipulate gastrointestinal microbiota to increase short-chain fatty acids (SCFAs) [116], and plays an animal productivity under diverse productive settings. important role in breaking down complex molecules to simpler compounds that are essential for host growth 5 Examples of commercial phytochemicals [117]. SCFAs are absorbed by the host and used as an and their synergistic action with other feed energy source but also have a variety of distinct physi‑ additives ological effects. SCFAs are saturated aliphatic organic 5.1 Tannins in animal husbandry acids that consist of 1–6 carbons of which acetate, pro‑ Tannins are present in many feeds such as fodder leg‑ pionate and butyrate are the most abundant (≥95%). umes, browse leaves and fruits. Although the structure Although Bacteroides generates acetate and propi‑ of tannins are chemically diverse, they have one unifying onate, its ability to produce butyrate has not been property: tannins bind proteins. During the last 30 years, reported. Order Clostridiales are generally known tannins have been successfully used in animal production as important contributors to short-chain fatty acid to improve health and productivity, and several prod‑ (SCFA) metabolism [86] because it contains a variety ucts based on blends of particular amounts of hydrolys‑ of bacterial families, among which Ruminococcaceae able (predominantly chestnut) and condensed (mostly and Lachnospiraceae are capable of fermenting various quebracho) tannins were developed to take advantage of substrates to butyrate. Feed tannin supplementation the benefts of each tannin in livestock. Tese products of chickens decreases the abundance of Bacteroides, are being used in many countries to improve quality and which could reduce acetate and propionate produc‑ production of milk, meat and eggs. In poultry, a blend tion. However, it would be compensated by an increase of tannins can be added to feed at a fnal concentration in Clostridiales, particularly Ruminococcaceae, with of 0.5–1 kg/tonne, both in pre-mix or directly into feed, a possible increase in butyrate production [96]. Con‑ to obtain several benefts including reduction of mortal‑ cordantly, Masek et al. [118] have reported a global ity rate, improvement of feed efciency, weight gain and increase in SCFA production in poultry treated with intestinal health, reduction of NE and foot-pad lesions, tannic acid. and increased feces consistency and litter quality of com‑ Lactic acid bacteria, which are usually associated mercial settings. Te selected blend of tannins added to with enhanced gut health and productivity, are inter‑ the diet stabilizes and increases feed intake according to esting. It was reported that cecal microbiota contained reduction of taste variation by changes in feed formula‑ lower proportions of Lactobacillus in AGP-fed chick‑ tion [126], and reduces feed stress by improving the fa‑ ens, compared with chickens in tannin and control voring characteristics. Te distinctive antispasmodic groups [119–121]. Lactic acid bacteria, especially Lac- efects of tannins that modulate gut motility [127, 128], tobacillus strains, have been considered as probiotic with strong antibacterial efects on several pathogenic microorganisms because of their activities in reducing bacterial species and viruses [97, 129], as well as their enteric diseases and maintaining poultry health [122– toxins [97], are used to prevent and control enteric dis‑ 124]. The presence of Lactococcus spp. has been cor‑ eases, including several diarrheal diseases [130] and NE related with weight gain [125]. [96]. Reduction of enteric diseases, intestinal motility The inclusion of different AGPs in diet influences the and bacterial load, concurrently with an increase of feed diversity of gastrointestinal microbiota. These changes digestibility, produces a reduction of humidity in the lit‑ would probably be one of the most important driving ter, afecting directly animal health and welfare. It has forces resulting in efficiency improvement of animal become obvious when foot-pad disorders are observed in production. Similarly, the existing information clearly commercial farms, dietary tannins reduced up to 50% of shows a significant alteration in the relative abun‑ the animals with lesions, and up to 20% reduction of ani‑ dance of specific bacterial populations by some phyto‑ mals with the most severe lesions. chemicals in the gut of domestic animals (13). These Tese blend of tannins are also being used efca‑ phytochemicals added to feed are also connected with ciously to reduce the incidence of sub-clinical NE, and a higher productivity parameters. Therefore, these nat‑ slightly diferent blend is able to strongly reduce intesti‑ ural compounds are able not only to improve animal nal lesions in chickens on farms with a history of severe health and welfare directly, but also to modulate gas‑ NE outbreaks. In experimental conditions, the tannin trointestinal microbiota and increase the impact on blend is able to reduce the most severe lesions as well Lillehoj et al. Vet Res (2018) 49:76 Page 12 of 18

as the number of animals with lesions. Tis result is also of tannin also reduced fecal moisture, resulting in better observed in commercial farms of diferent European, fecal consistency. American and Asian countries where NE is a problem According to Rivera-Mendez et al. [137], the addition to diferent degrees. As an example, an integrated com‑ of up to 0.2% of a blend of tannin to steers during the pany in Brazil with a persistent history of sub-clinical NE feedlot fnishing phase increased average daily gain by started using the tannin product in 2015 and reduced the 6.5%. Body weight in young animals was improved up to number of animals with lesions by 10%, improving pro‑ 7% in commercial conditions before the breeding period ductivity by almost 3% (Dr Joao Battista Lancini, personal [107, 138]. Similarly, DM intake tended to increase with communication). level of tannin. Tannin supplementation increased gain A comparative analysis of AGPs versus tannin blend efciency (5.5%) and dietary net energy (3.2%). Tese use in feed was carried out in a commercial trial in results have been also observed in commercial feedlot Argentina over a period of 13 months (5 cycles) in a poul‑ fnishing settings. Te analysis of 15 diferent trials in try farm of ~200 000 animals. Te farm was divided into North America between 2010 and 2013 using tannins at six barns under regular commercial feed; three were fed 0.25%, with or without antibiotics or ionophores in feed, with AGPs in feed and three with 0.1% blend of tannins in showed an average daily gain of 9.2% and gain efciency feed but without AGPs. Greater improvements in intesti‑ of 5.07% compared to non-tannin controls [139, 140]. nal health, microbiological quality and humidity of litters, Similar results have been observed in feedlots in other mortality rate, undigested feed, foot-pad lesions, and parts of the world, including large beef producers in Bra‑ weight gain were observed in the animals treated with zil [141, 142] and Argentina [136]. tannins versus antibiotics. Analysis of the results showed In conclusion, the addition of low-dose tannins to a positive diference of almost 10 points for the Produc‑ ruminant diets in intensive fattening is an available tion Efciency Factor for the blend of tannins against tool to increase nutrient use efciency, improving daily AGPs in feed, showing the benefts of using these blend weight gain and feed conversion, through diferent met‑ of tannins during diferent weather conditions through‑ abolic mechanisms. Te estimated level of animal feed out the year [131]. Tannins added in feed to improve pro‑ supplemented with tannins produced in the world in ductivity in combination with other products, including 2016 was 15 000 000 tonnes, refecting the acceptance EOs, organic acids, probiotics and AGPs, have been used of tannins as an important tool in animal husbandry. frequently by diferent companies in several countries Te available scientifc information about mechanism of with signifcant positive results (Dr Javier Quintar and Dr action, the observed animal response and the accumu‑ Joao Battista Lancini, personal communication). lated experience in the use of tannins as feed additive In cattle, historically low doses of quebracho and chest‑ confrms that tannins are a valuable alternative to com‑ nut tannins have been used in feed by many producers plement or replace the use of AGPs in industrial livestock around the world to improve bypass protein from rumen production. degradation. Rumen bypass protein is one of the strate‑ gies to increase the amount of protein that enters abo‑ 5.2 Synergistic action of phytochemicals with other masum and hence increases ruminant productivity. Te feed additive antibiotic alternatives for commercial reduction in protein degradation in the rumen may occur products by the formation of a reversible tannin–protein complex Designing an antibiotic alternative to address several in the rumen pH and/or the modulation of rumen micro‑ components of gut health may work better than using a biota. Te addition of such tannins to a diet reduces the single approach to reduce negative consequences of gut fermentability of protein nitrogen in the rumen [132]. damage caused by complex etiologies such as those that Consequently, the fow of dietary amino acids into the cause diseases such as NE. C. perfringens produces sev‑ duodenum of ruminants could be increased, as well as eral exotoxins, including α-toxin and NE toxin B (NetB), the total duodenal amino acid fow if ammonia nitrogen that disrupt the intestinal epithelium, causing necrotizing requirements for microbes could be met by supplementa‑ lesions that constitute the characteristic sign of NE [21, tion of urea or ammonia salts. 143]. In addition, added tannins are also used to prevent aci‑ For complex disease like NE, it takes a multi-fac‑ dosis and bloating [133], modulate rumen microbiome eted approach to decrease the efects of disease on gut to improve feed utilization [130], and reduce methane health. For example, a commercial product ­Varium® was emissions [134] and nitrogen excretion [135]. A par‑ designed to improve barrier function by removing patho‑ ticular tannin mix added in feed was able to reduce liver gens by agglutination, removing biotoxins via adsorption, abscesses in beef cattle by >80% [136]. Supplementation priming immune development, and providing energy to the enterocytes [144]. ­Varium® has been tested in vitro Lillehoj et al. Vet Res (2018) 49:76 Page 13 of 18

for its ability to bind biotoxins of pathogenic bacteria (i.e. that most people equate the phrase with something not C. perfringens and E. coli) such as α-toxin, NetB toxin, termed an antibiotic that can be substituted for low level lipopolysaccharide, heat-labile toxin and Shiga-like type feeding of broad-spectrum antibiotics used to promote 2 toxin. Te binding of these toxins was dose dependent, growth in livestock. Te reason there is a need for alter‑ with the exception of NetB toxin, which was bound 100% natives to AGP is the recognition that the practice can across the doses tested. lead to development of infective bacteria that are resist‑ Two large broiler trials have been conducted to test the ant to many of the current antibiotics available to human hypothesis that CaMM, or its blends with other materi‑ medicine. Te rising incidence of superbugs globally and als (e.g. fermentable fbers, organic acids, and/or phy‑ the rising human deaths from multiple drug-resistant tonutrients) could improve gut health and decrease the bacteria have alerted WHO, CDC and UN to release negative efects of avian NE. Te two trials evaluated strict action plans on reducing the use of antibiotics in CaMM-based dietary products on growth performance, animal production. clinical signs, immunopathology, and cytokine responses Regardless of which side of the argument over whether of young broilers using disease challenge models with AGP use in animals is contributing to the problem of avian NE [144]. When tested in unchallenged birds, resistant bacteria in humans you are on, the sociopoliti‑ Varium exerted an efect similar to an in-feed AGP on cal momentum has created a marketing opportunity for body weight, feed intake, and FCR. Chickens fed a diet selling meat from animals claimed to have never received supplemented with CaMM plus a fermentable fber and antibiotics during production. Tis in turn creates a mar‑ an organic acid showed increased body weight gain, ket for products that can provide the beneft of AGPs but reduced gut lesions, and increased serum antibody levels not be antibiotics used in human medicine, or sometimes to C. perfringens α-toxin and NetB toxin compared with any antibiotic at all. Te alternative to antibiotics market chickens fed the basal diet alone. Levels of transcripts for is growing rapidly and attracting interest from compa‑ infammatory cytokines such as IL-1β, IL-6, inducible nies and organizations of all sizes and capabilities. Tis is NO synthase, and TNFSF15 were signifcantly altered in evident from the need for a meeting such as this and the the intestine and spleen of CaMM-supplemented chick‑ plethora of products marketed, with or without credible ens compared with unsupplemented controls [144]. data, to be alternatives to AGPs. Although the banning of In Trial 2, Cobb/Cobb chickens were fed an unsupple‑ AGPs has accelerated over the last few years, the search mented diet or a diet supplemented with CaMM; each for alternatives started in earnest following the ban in the with a fermentable fber and an organic acid, and co- EU of avoparcin in 1997. infected with E. maxima and C. perfringens under sub‑ Te most important development in the search for clinical infection conditions to elicit NE. Compared with credible alternatives is the increasing understanding in unsupplemented controls, broilers fed with CaMM plus a both human and veterinary medicine that the gastroin‑ fermentable fber and an organic acid showed increased testinal tract is more than a nutrient-absorbing organ, body weight gain, reduced FCR, mortality, and intestinal but in fact is fundamental to health and development of lesions, compared with chickens fed an unsupplemented humans and animals. Te scientifc advancement in our diet. understanding of the importance of the gut environ‑ Based on both broiler trials, it is recommended that ment and its barrier function in health provide a way to dietary supplementation of CaMM or CaMM plus a fer‑ develop products that can deliver the benefts of AGPs mentable fber and an organic acid is useful to decrease without causing an increase in the emergence of antibi‑ negative efects of avian NE in the feld. Future studies otic-resistant bacteria. Tis can be accomplished by using are needed to characterize further the CaMM-regulated multiple technologies to maintain or strengthen gut bar‑ physiological and immunological mechanisms that are rier function. Scientifc principles should be applied to activated in response to avian NE. the development of products such that they provide reli‑ able positive benefts to the target animals. 5.3 Antibiotic alternatives: industry perspective In a recent survey, more than 70% of animal feed com‑ In general, there is a lack of consensus on what is meant panies showed interest in willingness to use somekind of by the phrase “antibiotic alternatives”. AGP use is a com‑ feed additive as antibiotic alternatives. However, there mon practice that has been around for >65 years in are still many challenges remaining with the most con‑ modern livestock production that to this day has no con‑ sistent concerns being consistency, safety and solid scien‑ sensus about its mechanism of action. Yet, most of the tifc proof. Tis is not surprising when you consider most technologies discussed here have proposed or known of the popular alternative products marketed today mod‑ mechanisms of action that involve inhibition, alteration ify the microbiota in some way to enrich benefcial bacte‑ or killing of one or more bacteria. In general, it appears ria. We are just learning what the desirable microbiota is Lillehoj et al. Vet Res (2018) 49:76 Page 14 of 18

and how it works in given animal, and we have even less antibiotics that promote growth in livestock. Antibiotic knowledge of the variations between diferent animals alternatives will be mainly used to replace AGPs whose and the normal daily and lifetime changes in diferent primary function is to decrease microbial populations ecosystem. So, it is likely that a product that can deliver and promote growth via many diferent modes of action consistent results will need to incorporate two or more that may include alteration and/or inhibition of micro‑ components that have complimentary and/or synergis‑ bial growth, decrease of infammation, enhancement tic mechanisms of action. In addition to the microbiota, of innate immunity, reduction of oxidative stress, and it will be necessary to understand clearly what impact improvement of gut integrity. Increasing marketing the product has on the gut barrier which comprises the opportunity for selling animal meat products claimed mucus layer, endothelial cells and attendant immunologi‑ to have never received an antibiotic (antibiotic-free, cal cells and structures associated with the gut wall. ABF; no antibiotics ever, NAE) has created a mar‑ Tis is a relatively new feld of research and as time ket for products that can provide the beneft of AGPs goes on, the industry, through application of good sci‑ without using antibiotics that are used therapeutically ence, will learn more. Tis will be both in the basic in human medicine. Te most important development understanding of the gut environment, including the in the search for credible alternatives to AGPs is the microbiota and the dynamic function of the gut barrier, new understanding in both humans and veterinary and how to manipulate these structures in individuals, animals that animals including humans are “superor‑ but as part of a population. Because it is new and there ganisms” that contain trillions of bacteria, with more are many unknowns, regulation of these products poses a than thousands of species, and that the gastrointesti‑ challenge in diferent regions of the world. What consti‑ nal tract is an intelligent sensory organ that not only tutes acceptable efcacy and what types of claims can be absorbs nutrients, but also communicates with the larg‑ supported are largely unknown. However, there is little est neuroendocrine system in the body. Tis new scien‑ doubt that use of the FDA drug approval process is not tifc knowledge in our understanding of the importance a viable option today. Perhaps as science defnes ways to of the gut environment and barrier function in health measure and test efcacy in a consistent manner across should guide fnding a future solution to develop novel several mechanisms of action, a regulatory pathway can products that can deliver the benefts of AGPs with‑ be established. Tere will need to be tolerance and fex‑ out causing an increase in the emergence of resistance. ibility in the approval process for these products or the For example, when we consider using phytochemicals market will be fooded by products with no proof of ef‑ as antibiotic alternatives, we need to consider: (1) dose cacy or safety. At a minimum, these products should have for immune versus bacteriostatic/cidal efect in target scientifc proof of efcacy in the target species for which animals; (2) variations in active compound in plants they are marketed. In vitro tests are insufcient to pro‑ and plant-derived products; (3) unexplored concurrent vide confdence that a product will work in an animal, efects of phytochemicals (antiviral and antineoplastic); let alone provide consistent value across a population of (4) target organs/tissues afected by phytochemicals; (5) animals. safety of phytochemical residues in humans; and (6) the long-term efect of using phytochemicals in animals on 6 Conclusions and future directions developing resistance. Since using phytochemicals as Increasing concerns about the increase of superbugs antibiotic alternatives in agricultural animals is a rela‑ and limited development of new drugs for livestock and tively new feld of research, regulation of these prod‑ humans necessitates the timely development of alterna‑ ucts poses a challenge. Tere is a timely need to provide tives to AGPs. With increasing availability of many dif‑ increased public funding for mechanistic research for ferent categories of antibiotic alternatives in the market phytochemicals that include standard measurements to for animal agriculture with various claims and efcacy, defne the efcacy in a consistent manner across several the industry needs to understand the mode of action regulatory pathways, to prevent false claims and yet associated with diferent types of antibiotic alterna‑ have fexibility in the approval process for proof of ef‑ tives and the kind of synergy that can be ofered by the cacy or safety for commercialization. Owing to the rise combinations of diferent antibiotic alternatives, espe‑ in consumer demand for livestock products from ABF cially for prevention and treatment of complex diseases production systems, scientists, regulatory agencies and such as necrotic enteritis. Furthermore, the defnition commercial partners need to work together to develop of the phrase antibiotic alternatives should be better efective antibiotic alternatives to improve performance defned, although this terminology is now an accepted and maintain optimal health of food animals. Using term to refer to non-antibiotic substances that can be optimal combinations of various alternatives coupled substituted for low-level feeding of broad-spectrum with good management and husbandry practices will Lillehoj et al. Vet Res (2018) 49:76 Page 15 of 18

be the key to maximizing performance and maintaining 5. Lee SH, Lillehoj HS, Jang SI, Kim DK, Ionescu C, Bravo D (2010) Efect of dietary curcuma, capsicum, and lentinus on enhancing local immunity animal productivity, while we move forward with the against Eimeria acervulina infection. J Poult Sci 47:89–95 ultimate goal of reducing antibiotic use in the animal 6. Lee SH, Lillehoj HS, Jang SI, Lee KW, Park MS, Bravo D, Lillehoj EP industry. Further research is needed regarding under‑ (2011) Cinnamaldehyde enhances in vitro parameters of immunity and reduces in vivo infection against avian coccidiosis. Br J Nutr standing their mechanism of action, identifying means 106:862–869 to standardize the efects, improving delivery methods 7. Lee SH, Lillehoj HS, Jang SI, Lillehoj EP, Min W, Bravo DM (2013) Dietary (e.g. microencapsulation) for site-targeted delivery, and supplementation of young broiler chickens with Capsicum and turmeric oleoresins increases resistance to necrotic enteritis. Br J Nutr increasing their in vivo efcacy in farm settings. 110:840–847 8. Kim DK, Lillehoj HS, Lee SH, Jang SI, Lillehoj EP, Bravo D (2013) Dietary Abbreviations Curcuma longa enhances resistance against Eimeria maxima and ABF: antibiotic-free; AGPs: antibiotic growth promoters; DM: dry matter; EOs: Eimeria tenella infections in chickens. Poult Sci 92:2635–2643 essential oils; FDA: Food and Drug Administration; HMG-CoA: hydroxymeth‑ 9. Lee SH, Lillehoj HS, Hong YH, Jang SI, Lillehoj EP, Ionescu C, Mazura‑ ylglutaryl coenzyme A; IFAs: in-feed antibiotics; IFN: interferon; IL: interleukin; nok L, Bravo D (2010) In vitro efects of plant and mushroom extracts LPS: lipopolysaccharide; NAE: no antibiotics ever; NE: necrotic enteritis; NO: on immunological function of chicken lymphocytes and mac‑ nitric oxide; OIE: World Organization for Animal Health; OUT: operational rophages. Br Poult Sci 51:213–221 taxonomic units; PTS: propyl thiosulfnate; PTSO: propyl thiosulfnate oxide; 10. Lee SH, Lillehoj HS, Jang SI, Lee KW, Bravo D, Lillehoj EP (2011) Efects SCFA: short-chain fatty acid; SFBs: segmented flamentous bacteria; TNFSF15: of dietary supplementation with phytonutrients on vaccine-stimu‑ TNF superfamily member 15; VFAs: volatile fatty acids; VFD: Veterinary Feed lated immunity against infection with Eimeria tenella. Vet Parasitol Directive. 181:97–105 11. Bravo D, Pirgozliev V, Rose SP (2014) A mixture of carvacrol, cinna‑ maldehyde, and capsicum oleoresin improves energy utilization and Competing interests growth performance of broiler chickens fed maize-based diet. J Anim The authors declare that they have no competing interests. Sci 92:1531–1536 12. Bravo D, Ionescu C (2008) Meta-analysis of the efect of a mixture of Authors’ contributions carvacrol, cinnamaldehyde and capsicum oleoresin in broilers. Poult All the authors participate in drafting the article. All authors read and Sci 87:75 approved the fnal manuscript. 13. Kim JE, Lillehoj HS, Hong YH, Kim GB, Lee SH, Lillehoj EP, Bravo DM (2015) Dietary Capsicum and Curcuma longa oleoresins increase Acknowledgements intestinal microbiome and necrotic enteritis in three commercial The authors thank members of the ATA Symposium Scientifc and Organizing broiler breeds. Res Vet Sci 102:150–158 Committees, staf of the World Organization for Animal Health (OIE) and to the 14. Settle T, Leonard SS, Falkenstein E, Fix N, Van Dyke K, Klandorf H Agricultural Research Service (ARS) of the USDA. (2014) Efects of a phytogenic feed additive versus an antibiotic feed additive on oxidative stress in broiler chicks and a possible mecha‑ Author details nism determined by electron spin resonance. Int J Poult Sci 13:62 1 Animal Biosciences and Biotechnology Laboratory, Agricultural Research 15. Kim DK, Lillehoj HS, Lee SH, Jang SI, Bravo D (2010) High-throughput Service, US Department of Agriculture, Beltsville, MD 20705, USA. 2 University gene expression analysis of intestinal intraepithelial lymphocytes of California, Davis, CA 95616, USA. 3 Animal Nutrition and Welfare Service, Uni‑ after oral feeding of carvacrol, cinnamaldehyde, or Capsicum oleo‑ versitat Autònoma de Barcelona, 08193 Bellaterra, Spain. 4 Instituto de Patobi‑ resin. Poult Sci 89:68–81 ología, Centro Nacional de Investigaciones Agropecuarias, Instituto Nacional 16. Kim DK, Lillehoj HS, Lee SH, Lillehoj EP, Bravo D (2013) Improved de Tecnología Agropecuaria, Calle Las Cabañas y Los Reseros s/n, Casilla de resistance to Eimeria acervulina infection in chickens due to dietary Correo 25, Castelar, 1712 Buenos Aires, Argentina. 5 Amlan International, supplementation with garlic metabolites. Br J Nutr 109:76–88 Chicago, IL 60611, USA. 6 National Program Staf‑Animal Health, Agricultural 17. 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