Phytogenic Feed Additives in Poultry: Achievements, Prospective and Challenges

Simple Summary Plant secondary metabolites and essential oils also known as phytogenics are biologically active compounds that have recently attracted increased interest as feed additives in poultry production, due to their ability to promote feed efficiency by enhancing the production of digestive secretions and nutrient absorption, reduce pathogenic load in the gut, exert antioxidant properties and decrease the microbial burden on the animal’s immune status. However, the mechanisms are far from being fully elucidated. Better understanding the interaction of phytogenics with gastrointestinal function and health as well as other feed ingredients/additives is crucial to design potentially cost-effective blends. Abstract Phytogenic feed additives have been largely tested in poultry production with the aim to identify their effects on the gastrointestinal function and health, and their implications on the birds’ systemic health and welfare, the production efficiency of flocks, food safety, and environmental impact. These feed additives originating from plants, and consisting of herbs, spices, fruit, and other plant parts, include many different bioactive ingredients. Reviewing published documents about the supplementation of phytogenic feed additives reveals contradictory results regarding their effectiveness in poultry production. This indicates that more effort is still needed to determine the appropriate inclusion levels and fully elucidate their mode of actions. In this frame, this review aimed to sum up the current trends in the use of phytogenic feed additives in poultry with a special focus on their interaction with gut ecosystem, gut function, in vivo oxidative status and immune system as well as other feed additives, especially organic acids.


Introduction
Poultry production is undergoing a continuous challenge to develop management strategies to optimize chickens' efficiency while limiting food safety concerns. Traditionally, antimicrobials have been widely used for improving health and growth performance in poultry; however, the increased public awareness about the risk of developing crossresistance of pathogens to antibiotics has resulted in the gradual removal of antibiotics for therapeutic and prophylactic uses in food animals [1]. The shift away from antibiotic supplementation has resulted in a tremendous growth in research focusing on the implementation of effective alternative control methods, management and dietary amendments aiming to improve animal health, welfare, and productivity. A wide range of feed additives including a broad spectrum of essential oils and related compounds from botanical sources to organic acids [1,2], as well as probiotics and prebiotics [3], chemicals such as aldehydes [4], bacteriophages [5], zinc oxide [6], exogenous enzymes [7] and competitive exclusion products [8] have been used in animal production. Particularly, phytogenic feed additives the amount and composition of ingested food [11]. Therefore, it secretes gastrointestinal peptide hormones, such as gastrin, secretin and cholecystokinin in response to nutrient, neural or hormonal stimulation as well as by metabolic products of the gut microbiota. These peptide hormones are involved in the digestive and absorptive function of the GIT such as the regulation of gastric acid and pancreatic secretion, release of bile from the gall bladder and gut motor activity [9].
The diet is a main factor modulating the composition and the metabolic activity of the GIT microbiota [21]. In this regard, several feed additives have been developed focusing on enhancing immune response, reducing pathogen load in the GIT, promoting the colonization of the GIT with beneficial bacteria and stimulating digestion and absorption. The current review will discuss the effects of PFAs on the different components of GIT functionality.

Phytogenics as an Alternative to Antimicrobials in Poultry Feeding
A broad range of plants derived products may fall under the category of phytogenic feed additives. They may be classified either based on their origin (the part of the plant) into herbs (products from flowering, non-woody, and non-persistent plants from which leaves and flowers are used) and spices (non-leaf parts of plants, including seeds, fruits, bark or root with intensive taste or smell); or depending on the process used to derive the active ingredients as essential oils (EOs: volatile lipophilic substances obtained by cold extraction or by steam or alcohol distillation) and oleoresins (extracts derived by non-aqueous solvents). The bioactive components of PFAs are secondary metabolites being polyphenols the main group. Other bioactive compounds include terpenoids (monoterpenes, steroids. . . ), phenolics (tannins), glycosides, and alkaloids [21]. The composition and concentration of these bioactive substances may vary according to several factors including the plant, parts of the plant, geographical origin, harvesting season, climatic conditions, processing techniques such as extraction, distillation and stabilization as well as storage conditions [22,23].
In recent years, PFAs have attracted an increasing attention as natural alternative to antibiotic growth promoters (AGPs) in poultry production which can be included in feeds as dried, solid, and ground form, or as extracts (crude, concentrated and purified) [24]. A wide variety of herbs and spices (thyme, oregano, cinnamon, rosemary, marjoram, yarrow, garlic, ginger, green tea, black cumin, coriander, among others) as well as EOs (from thymol, carvacrol, cinnamaldehyde, garlic, anise, rosemary, citruses, clove, ginger) have been used in poultry, individually or mixed, for their potential application as AGP alternatives [24]. Although the repertoire mechanisms of action of PFAs is not fully elucidated in poultry, one of their primary mode of action is related to their antimicrobial effects which allow controlling potential pathogens [25]. The results obtained in some recent studies will be reported with more details in the current review. However, some authors reported no positive effects of PFAs inclusion [26]. This discrepancy may be attributed to several factors, including the inherent variability of the botanic composition, as well the variability of the animal scenarios, environmental, management and sanitary conditions (i.e., including the likely presence of a pathogen challenge). The technique of treatment (cold, steam distillation, extraction or maceration with non-aqueous solvents. . . ) has been also reported to change the active substances and related compounds in the final product [22].
Among PFAs, there is a rising interest in EOs for animal nutrition as some of these feed additives have been shown to possess a much higher biological activity compared to the raw material they were extracted from [27]. EOs are complex mixtures of volatile compounds, being mainly hydrocarbons (terpenes, sesquiterpenes), oxygenated compounds (alcohol, aldehydes, ketones) and a small percentage of non-volatile residues (paraffin, wax) [28]. Chemically, EOs consist fundamentally of two classes of compounds, the terpenes and phenylpropenes. Although, the effects of a mixture of EOs rely on the additive and synergy or antagonic effects of their components, 2 or 3 components may constitute up to 85% of the total mixture [29] and thus, contribute to its primary property [2]. In fact, thymol and carvacrol are the two main phenols which account for almost 80% of the EO of oregano and are the main contributors to its antibacterial and antioxidant activities. The compound pcymene is another dominant component of oregano EO [30]. Even though this component is not considered as an effective antimicrobial agent, it is a precursor for carvacrol which possesses higher preference for liposomal membranes, enabling carvacrol to be more easily transported into the cell [31].
EOs are perceived as growth promoters in poultry diets with strong antimicrobial and anticoccidial activities [2]. Thus, literature shows that growth promoting effects of EOs exist both abundantly and controversially, which makes it imperative to perform more in-depth research to understand the underlying mechanisms.

Effects of Phytogenic Feed Additives on Chickens
The effects of PFAs on the main metabolic and physiologic process like nutrient digestibility, intestinal microbiota, immunity, oxidant status and growth performance of broilers will be discussed with details below.

Effects of Dietary PFA Supplementation on Growth Performance
Several studies have been carried out using herbs, spices, and EOs and showed inconsistent results on chicken performance. Although some studies showed that PFAs have positive effects on body weight gain and FCR in chickens [32,33], others reported either an improved chicken body weight gain without affecting FCR [34,35] or an enhanced feed conversion rate associated to a lack of effects on body weight or feed intake [36,37]. This inconsistency may be explained by several factors such as the botanical source, the concentration and the duration of supply of the active compounds, the feed composition, and the experimental challenging conditions, animal age and health status [38].

Chickens Maintained under Non-Challenging Conditions
Detailed results of some recent studies evaluating the effects of PFAs on growth performance of birds maintained under non-challenging conditions are presented in Tables 1-3. Dietary supplementation of EOs containing menthol, anethol and eugenol [39] as well as carvacrol alone [40], or combined with either thymol [41][42][43] or thymol and limonene [44] has been shown to promote growth performance of broiler chickens. Similar positive effects on production performance were obtained by supplementing laying hens diets with thymol and cinnamaldehyde [39], star anise oil [45], Citrullus lanatus EO [46], tea tree EO [47] and peppermint oil [48]. Several other studies have also evaluated the effects of supplementing black cumin (Nigella sativa L.) seeds on broilers, quails and laying hens. Although some authors failed to find any effect by supplementing Japanese quails diets with black cumin [49], others reported improved growth performance in quails [50,51] broiler chickens [52] and laying hens [53,54]. These growth-promoting effects have been attributed to the presence of a large number of pharmacologically active compounds such as thymoquinone, dithymoquinone, thymohydroquinone, nigellone, melanthin, nigilline, nigelamine, damascenone, p-cymene and pinene and a variety of essential nutrients including vitamins A, B, C, D and E, as well as minerals such as magnesium, calcium, phosphorus, potassium, iron, cobalt, zinc and manganese [41,55]. Moreover, curcuminoids and lipophilic turmeric extract containing curcumin and turmerones; known for their gastroprotective and anti-inflammatory activities, showed positive effects on growth performance of slow-growing [56] and fast-growing broiler chickens [57], respectively. Similar growth-promoting effects were observed by supplementing broiler chickens by a PFA of Aerva lanata, Piper betle, Cynodon dactylon, and Piper nigrum [58], Pulicaria gnaphalodes powder [59], Achyranthes japonica extract [60], Boswellia serrata [61] and bioactive olive pomace extract from Olea europaea [62] as well as laying hens by fennel seeds or red pepper [53] a mixture of Punica granatum, Thymus vulgaris, and Allium sativum [63] and dietary Nettle (Urtica cannabina) [64]. However, egg weight, laying rate and FCR were not improved by dietary supplementation of either EO of star anise (Illicium verum Hook.f.) [45] or a mixture of 13.5% thymol and 4.5% cinnamaldehyde [65].

Chickens Maintained under Challenging Conditions
A large number of studies have been performed to investigate the positive effects of PFAs on broiler chickens subjected to challenges related to environmental conditions including heat stress, lipopolysaccharide (LPS) and pathogens such as Clostridium perfringens, Eimeria spp, Salmonella typhimurium, or Escherichia coli, among others. Results of some recent studies are illustrated in Tables 2 and 4.
Heat stress in chickens induces a tight junction disruption that may lead to increased gut permeability and eventually to a dysregulation of the body's homeostasis [66], and thereby poor nutrient absorption, increased secretion of electrolytes and water in gastrointestinal tract leading to compromised performance [67]. However, the adverse effects caused by heat stress on broiler growth performance have been shown to be alleviated by the supplementation of turmeric rhizome powder [68], enzymatically treated Artemisia annua [69], ginger [70] and curcumin [71]. In ovo injection of black cumin (Nigella sativa) extract improved post-hatch performance of thermally challenged broiler chickens during incubation [72]. Similarly, 59-week-old cold-stressed laying hens supplemented with oregano EO showed improved FCR and egg production from week 9 to 12 of a 12-week feeding trial [73].
Numerous studies have been also performed to discern the efficacy of PFAs in chickens subjected to coccidiosis and necrotic enteritis (NE) classified among the most significant diseases affecting the poultry industry, which have become more prominent in the wake of policies to reduce the use of antibiotics in animal production. Clostridium perfringenschallenged broilers supplemented with PFAs consisting of either benzo [c]phenanthridine alkaloids from Macleaya cordata, active component of carvacrol from oregano (4.95 g/100 g), cinnamaldehyde from cinnamon (2.97 g/100 g), and capsaicin from paprika (1.98 g/100 g), or EO of thyme and anise as leading active ingredients and other including oregano, carvacol, yucca extract and cinnamaldehyde exhibited similar growth performance as the control non-infected group [74]. Microcapsules with a blend of EOs (thyme, peppermint, savoury, and black pepper) at the dose of 0.5, 1, and 2 kg/ton in the C. perfringens-challenged broiler chickens results in raising final weight and total feed intake [75]. However, supplementation of EO containing 25% thymol and 25% carvacrol as active components, did not influence the growth performance during d 0 to 14 and tended to linearly reduce the FCR between 14 and 28 d of age of Clostridium perfringens challenged broilers [76]. Regarding coccidiosis, the supplementation of a cashew nut shell oil and commercial castor oil blend allowed a recovery in performance similar to that observed with birds receiving the ionophore monensin during the accumulated experimental period (1 to 42 d) [77]. Hussein et al. reported that combinations of peppermint, chamomile and prebiotic yeast cell wall were as effective as salinomycin in preventing the decline in the weight gain and FCR performance of coccidiosis-challenged broilers [78]. However, the effects of dietary Yucca-derived saponin supplementation on growth performance seem to be dependent on broiler health status. Although Su et al. [79] reported that saponin supplementation via an extract from Y. schidigera serves as an effective growth promoter in non-challenged broilers, Oelshlager et al. [80] found no significant influence of Yucca extract on growth responses of broilers during a mixed coccidian challenge. This suggests a reduction in the bioefficacy of saponins when used during an immune challenge [80].
Similarly, a 20-day experiment showed that dietary curcumin supplementation from day 12 to 20 failed to positively affect growth performance of broiler chickens challenged with Eimeria species on day 14 of age [81]. These authors attributed the lack of effect to the short period of supplementation and suggested that it might be beneficial if curcumin is fed in broilers for a 42-d period. This suggestion was based on the results of Rajput et al. [82], showing that birds fed curcumin-supplemented diets for 42 d exhibited significant increase in the BW and feed efficiency during the finisher stage (22-42 d), whereas no significant difference in growth performance was observed during the starter phase (0-21 d).
PFAs supplementation has been also shown to alleviate the effects on the broilers growth performance of other pathogens, such as Achyranthes bidentate under Escherichia coli challenge [83], resveratrol under Escherichia coli challenge [84], a PFA consisting of various nutritional acids and four different alkaloids obtained from special plants under Salmonella typhimurium challenge [85], and Allium hookeri roots in LPS-induced young broiler chickens.

Effects of Dietary PFA Supplementation on Digestibility
Evaluating digestibility is important as it directly contributes to the animal feed efficiency. However, improving the digestibility is crucial not only for better feed efficiency but also to reduce the amount of undigested feed in the gut, which may favour the occurrence of intestinal imbalances. These imbalances may lead to inflammatory processes and accelerated turnover of intestinal tissue, which results in poorer performance.
Numerous studies have been carried out to study the effects of PFAs inclusion on nutrient digestibility in broiler chickens, laying hens as well as ducks and showed inconsistent results.
The use of either extracts from olive leaves rich in polyphenols [110] or a bioactive olive pomace extract from Olea europaea [62] failed to enhance nutrient apparent total tract digestibility (ATTD) coefficients, or apparent ileal digestibility (AID) of dry matter (DM), organic matter (OM), ether extract (EE), and gross energy (GE), respectively. However, the use of EOs such as a blend of carvacrol combined with either cinnamaldehyde and capsicum oleoresin [111] or thymol and limonene in encapsulated forms [44] increased fat digestibility (FD), the AID of crude protein (CP), phosphorus and cysteine. Similarly, a combination of over 30 essential oils and phytogenic compounds increased the digestibility of DM, CP and EE [112].
In laying hens, dietary supplementation of peppermint oil at 0, 74, 148, 222, and 296 mg/kg linearly increased digestibility of CP, EE, and phosphorus from 32 to 44 weeks of age [48], while 100 mg/kg of EOs including thymol 13.5% and cinnamaldehyde 4.5% as major active components significantly increased protein and fat digestibility from 54 to 65 weeks of age [65].
An increased nutrient digestibility was also obtained by supplementing meat-type ducks fed high nutrient density diets by a phytogenic blend containing quillaja, anise, and thyme [113]. Similar results were obtained in broiler chickens supplemented with an EO blend, a quillaja saponin blend, or a combination of both phytogenic preparations [114] or a PFA of Aerva lanata, Piper betle, Cynodon dactylon, and Piper nigrum [58]. These authors attributed the digestion-stimulating properties of the PFA basically to piperine, which has been previously reported to stimulate digestion and increase absorption of selenium, vitamin B complex, β carotene, and other nutrients.
Possible mechanisms behind improved nutrient digestibility by PFAs supplementation could be attributed to the ability of these feed additives to stimulate appetite, saliva secretion, intestinal mucus production, bile acid secretion, and activity of digestive enzymes such as trypsin and amylase as well as to positively affect the intestinal morphology [58] or to possess an overlapping mode of action including local effects at the intestinal border and systemic alterations of macronutrient metabolism by these feed additives [114].

Effects of Dietary PFA Supplementation on Intestinal Microbiota
It is well-known that farm animal performance is directly linked with gut function and health, which is determined by the continuous interaction among diet, intestinal integrity, gut microbiome and the immune system of chickens. Keeping in view the importance of intestinal microbiome [115], several authors studied the effects of PFAs on the microbiota composition ( Table 5). The results showed that these effects are both phytogenic composition and inclusion level dependent [116,117], and can range from neutral with no effect [117][118][119] up to beneficial [120][121][122][123][124][125].
The supplementation of a PFA containing the carvacrol as main active compound [124] was shown to modulate the intestinal microbiota more at cecal rather than ileal level by increasing cecal mucosa-associated levels of Bacteroides spp., Clostridium cluster IV, and Clostridium cluster XIVa. This increase could be considered beneficial as Clostridia are not only dominant in ceca [126], but they also contribute to the maintenance of overall gut function, especially through butyrate production [127]. Deinococcus, Bacillaceae and Caulobacteriales were increased in ileal digesta of laying hens fed on a dietary supplementation of EOs, promoting an increase of digestive enzyme activity leading to improved feed utilization efficiency [128]. Moreover, although some authors reported a decrease of the relative abundance of beneficial commensal bacteria such as Lactobacillus by plant extract supplementation [43,129], several others found Clostridiales and/or Lactobacillales to be higher in broilers supplemented with EOs of carvacrol and thymol [130,131] or thymol, eugenol and piperine [121]. More precisely, addition of thymol and carvacrol to the diet changes the host ileum microbial population dynamics by increasing the abundance of L. crispatus and L. agilis, and decreasing L. salivarius and L. johnsonii [131]. L. crispatus is known to be a rod-shaped species of the genus Lactobacillus and is a hydrogen peroxideproducing beneficial microbial species that plays a key role in the protection of the host from infection [132]. A study conducted by Gudiña et al. [133] revealed that a biosurfactant produced by a L. agilis strain showed considerable anti-adhesive activity against S. aureus, as well as antimicrobial activity against P. aeruginosa, S. aureus and S. agalactiae. Green tea and pomegranate have also been proven to modulate the intestinal microbiota [122,134], especially by promoting beneficial bacteria in the intestinal tract [125,135]. In a recent study conducted by Perricone et al. [123], broilers receiving a plant extract composed of green tea leaves (Camellia sinensis) and pomegranate rinds (Punica granatum) promoted greater relative abundance of lactic acid bacteria compared to the control group. These results are of particular interest because lactic acid bacteria are recognized to positively affect the intestine by regulating the composition of intestinal microflora, developing intestinal immunity and promoting gut health [136].  ↑: increased; ↓: decreased; =: equal.

Effects of Dietary PFA Supplementation on Immunity
An increasing number of studies have shown that health-promoting activities of phytochemicals are attributed to their ability to improve host defence against microbial infection [138]. The immune activating properties of several phytochemicals such as dandelion (Taraxacum officinale), mustard (Brassica juncea) and safflower (Carthamus tinctorius) [139] as well as thistle (Silybum marianum), turmeric (Curcuma longa), reishi mushroom (Ganoderma lucidum), and shiitake mushroom (Lentinus edodes) [140] have been evaluated in vitro using avian lymphocytes and macrophages. In both studies, all extracts inhibited tumour cell growth and stimulated the innate immunity in poultry. These results were further confirmed by several in vivo trials, which are giving increasing evidence that through interactions with immune system, PFAs can modulate immune responses through various mechanisms. Some of the most recent studies are synthesized in Table 6. Dietary immunomodulation is a key to enhance the productivity and immune system integrity of farm animals raised under the absence of antibiotics [141]. One of those mechanisms is the modulation of the expression of the cytokines playing a key role in both the adaptive and the innate immune system [142,143].  A study conducted by Pirgozliev et al. [86] demonstrated that broilers under nonchallenging conditions, supplemented by a commercial blend comprising 5% carvacrol, 3% cinnamaldehyde, and 2% capsicum oleoresin showed downregulated IFN-γ and IL-6 cytokines indicating a lower inflammation level than those in the other groups.
In broilers under challenging conditions such as necrotic enteritis, Lee et al. [150] showed that a mixture of Capsicum and turmeric oleoresins reduced intestinal IL-8, lipopolysaccharide-induced TNF-α factor (LITAF), IL-17A and IL-17F mRNA levels. Similarly, the expression of pro-inflammatory cytokines was reduced by the supplementation of Allium hookeri in LPS-induced young broiler chickens [148] and thyme powder in broilers without any challenge [87]. Nonetheless, a blend of cashew nut shell liquid (CNSL) and castor oil modulated the inflammatory response of broiler chickens against Eimeria spp. by increasing gene expression of TNF-α, IL-6 and IFN-γ and reducing expression of IL-1 and COX-2, one week post-infection [146] These authors concluded that although inflammation is a highly undesirable phenomenon owing to its costly effects on animal production efficiency, this increased inflammatory response observed in challenged birds treated with CNSL-castor oil was necessary to help the immune system to effectively fight against coccidiosis and other pathogenic bacteria to prevent intestinal dysbiosis.
Other authors have rather evaluated the levels of SIgA, IgM, and IgG, three major classes of immunoglobulin in chickens that play key roles in the maintenance of immunity [151]. SIgA is involved in the protection and homeostatic regulation of intestinal mucosal epithelia by limiting the access of numerous microorganisms and mucosal antigens, while IgG directly contributes to an immune response including neutralization of toxins and viruses. A decrease of SIgA and IgG observed in the jejunum mucosa of heat-stressed broilers was counteracted by the supplementation of enzymatically treated Artemisia annua [69]. Similarly, an increase of IgG was observed by the supplementation of thyme powder [87] and EOs of oregano [88] in broilers raised under normal conditions or by the supplementation of resveratrol in chickens challenged with Escherichia coli [84].
The effect of PFAs, especially EOs, on the immune response to some viral vaccines in broiler chickens was also evaluated in several studies. A mixture containing oregano oil (50 g), carvacrol (10 g), thyme oil (33.33 g), eucalyptus oil (50 g), thymol (5 g), eucalyptol (10 g), and acacia (Arabic gum) surfactant (27 g) supplemented through the drinking water at a dose of 0.5 mL/L showed an immune-stimulating response to Newcastle disease (ND) and infectious bursal disease (IBD) vaccines, antiviral effect against ND virus, especially if administered before the challenge [145].
The supplementation of black cumin (Nigella sativa) combined with Echinacea enhanced the immune response after AI-H9N2 vaccination and reduced the pathogenicity of infection in dexamethasone-stressed chickens [156]. The immune-modulating effects of black cumin may be attributed to pharmacologically active constituents, such as thymol, nigellidine, nigellimine, thymoquinone, dithymoquinone and thymohydroquinone [157] which are able to induce pharmacological effects against antigenic challenge [158].

Effects of Dietary PFA Supplementation on Blood Biochemical Parameters and Oxidant Status
Several authors studied the effect of PFA supplementation on the serum biochemical indicators that help to display the nutrient's metabolism and body physiological state [159]. Serum lipid parameters have been shown to be reduced in broiler chickens by the supplementation of lavender EO (cholesterol and LDL-C; [160]), Pulicaria gnaphalodes powder (cholesterol and triglycerides; [59]) and a mixture of oregano, anise and citrus EOs (cholesterol; [161]). Serum cholesterol has been also reduced in laying hens supplemented by EOs [162], either black cumin or hot red pepper [53], peppermint oil [48] or nettle Urtica cannabina [64].
The mechanisms explaining the hypocholesterolemic effect of PFAs may be associated to the reduced activity of enzymes involved in lipid metabolism including 3-hydroxy-3methylglutaryl-CoA (HMG-CoA) reductase (enzyme associated with cholesterol synthesis), cholesterol-7 hydroxylase fatty acid synthase and pentose phosphate pathway [147,163].
The supplementation of PFAs has been also shown to improve the antioxidant status of broilers [164][165][166]. A recent study performed by Paraskeuas et al. [39] revealed that a mixture of menthol, anethol and eugenol increased blood plasma total antioxidant capacity (TAC) in a linear pattern, corroborating the results previously obtained by other authors [167,168]. The activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), enzymes considered as one of the defensive mechanism of the body against the oxidative stress [169], was increased by supplementing broilers by lavender EO [160] and laying hens by star anise oil [45] and grape pomace [101]. Malondialdehyde (MDA) levels were also decreased in laying hens supplemented by grape pomace [98] and star anise oil [45].

Effects of Dietary PFA Supplementation on Meat, Internal and External Egg Quality
Benefits of dietary PFA supplementation on the quality and shelf-life of meat products are still ambiguous. Although the supplementation of a PFA based on EOs of carvacrol, thymol and cinnamic aldehyde was unable to prevent broiler meat lipid peroxidation caused by freezing temperatures [42], other authors reported increased antioxidant levels and reduction of lipid peroxidation by Nigella sativa seeds [52], turmeric [57], curcuminoids [56] as well as herbal components containing curcumin, carvacrol, thymol and cinnamaldehyde [144]. Manipulating lipid peroxidation has been shown to be, in part, achieved through modulating the profile of meat fatty acids by PFAs supplementation. Total saturated fatty acid (SFA) levels were reduced and monounsaturated/polyunsaturated fatty acid (MUFA/PUFA) levels were increased by PFAs supplementation [56,144,170,171]. Particularly, SFA such as lauric, stearic, myristic and palmitic acid are undesirable due to their hypercholesterolemic properties in the form of LDL [144,170]. On the contrary, omega 3 and 6 fatty acids play key roles in human nutrition¸being precursors of principal molecules involved in the regulation of the cardiovascular and immune system including eicosanoids, prostaglandins, leukotrienes and thromboxanes [144].
External and internal egg quality has been also shown to be influenced by PFAs supplementation. Eggshell thickness was increased by supplementation of herbal EOs mixture [162], peppermint oil [48] and nettle Urtica cannabina [64]. A possible mechanism behind these positive effects is the ability of PFAs such as EOs to improve uterine health and increase calcium storage as well as pancreatic secretions, resulting in the enhancement of nutrient digestion and consequently the improvement in eggshell and egg quality [48].
Regarding internal quality, the supplementation of PFAs such as black cumin or red pepper [53], star anise (Illicium verum Hook.f.) EO [45] grape pomace flour [101] and curcumin [100] has been shown to reduce egg lipid peroxidation and increase its antioxidant levels, thereby, generating internal stability of the stored eggs and contributing to extending egg shelf life. The Haugh unit score, known as an indicator of egg freshness and is related to shelf life, has been also shown to be increased by PFAs supplementation [46,48,63]. Similar to meat, egg yolk cholesterol content has been shown to be reduced by PFAs supplementation [48,64]. Although the egg yolks of laying hens supplemented with Citrullus lanatus EOs showed increased total PUFA and n-6 fatty acids and reduced n-3 fatty acids [46], nettle Urtica cannabina supplementation increased total n-3 PUFA concentration while reducing the ratio of n-6/n-3 [64].

Challenges of Using PFAs in Animal Nutrition
Performing systematic and comprehensive studies evaluating the efficacy and safety of PFAs is still difficult due to their complex composition [172]. In addition, inconsistency in the obtained results may be attributed to several factors related either to the enormous variability per se of PFA, including source and bioactive compounds of the PFAs which may depend on the plant, botanical origin growing locations, manufacturing methods, the storage conditions, and the effective dose; or to the environmental conditions, management and rearing conditions (challenge vs. no challenge and differences in the underlying microbial challenge if applied, age, genetics. . . [22,172]. Some authors also reported that the appropriate minimum inhibitory concentration (MIC) for most phytogenic compounds is higher than the level considered as cost-effective [172]. On the other hand, various phytogenic compounds such as EOs can evaporate rapidly due to their volatile and reactive nature, resulting in largely varied concentrations in the final feed additive. Their effectiveness in animals may also be affected by several conditions during production processes and storage. Thus, ensuring their stability presents a difficulty, as do maintaining their biological activity and masking their strong odour [173]. Moreover, mutual interactions with other substance from feed matrix have been reported such as lower biological effects of PFA present in fibrous diets or high protein diets [174]. Several phytogenic compounds have been also shown to be largely absorbed in the upper GIT, meaning that without proper protection, the majority would not reach the lower gut where they would exert their major functions. In this context, a study conducted by Hafeez et al. [44] showed that the benefits of supplementing the broiler diet with a mixture of encapsulated EOs were higher than the tested PFA in powdered, non-protected form.
Therefore, novel delivery technologies have been developed to protect PFAs from the degradation and oxidation process during feed processing and storage, ease the handling, allow a slower release and target the lower GIT [173]. Among these techniques, microencapsulation is gaining an increasing interest where various carrier types including polymer-based particles such as polysaccharide-protein carriers and lipid-based particles such as vegetable oils and liposomes were tested. The advantages and disadvantages of both carrier materials from the encapsulation efficiency, loading capacity, and release kinetics viewpoints were previously discussed in the literature. Although advantages of polysaccharide-protein carriers include their mechanical and thermal stabilities, nutritional quality, low cost, and easy preparation procedure, they present low encapsulation efficiency, loading capacity, and release efficiency in small intestine [175]. As for lipid-based particles, they are characterized by high encapsulation efficiency, loading capacity, and release efficiency in the small intestine. However, their disadvantages include low mechanical and thermal stabilities [173]. Moreover, liposomes cannot be used for large scale production owing to complex preparation procedures, reduced production capacity, and higher cost [176].
Despite some studies showed some positive effects on chickens by using single EO, several others have rather chosen the combination of various EOs and their isolated components to take advantages from their synergistic effects [2,177]. Indeed, synergistic interactions are of great importance because they enhance the antimicrobial and antioxidant activity by maximizing the efficiencies of the combined agents in the best possible manner which results in several fold reduction in the required doses of EOs applied in situ and thereby lowering their organoleptic impacts. Interestingly, blends containing hydrocarbons and phenylpropanoids (e.g., cinnamaldehyde, eugenol, carvacrol, and capsicum oleoresin) in combination with other components were reported to enhance the bioactivities of these mixtures [178]. A special attention was placed on the interaction of phenolic monoterpenes (thymol, carvacrol) and phenylpropanoids (eugenol) with other groups of components, particularly with other phenols, phenylpropanoids and monoterpenes alcohols, whereas monoterpenes and sesquiterpenes hydrocarbons were used to a lesser extent [178]. Moreover, combining phenolics with monoterpenes alcohols has been reported to produce synergistic effects on several microorganisms, in particular, the combination of phenolics (thymol with carvacrol, and both components with eugenol) were synergistically active against Escherichia coli strains [178]. In ruminants, these combinations were the most effective dietary supplementation options that showed ruminal antimicrobial advantages to modulate the ruminal fermentation pathways [179,180].

Prospective of PFAs in Animal Nutrition: Combination of EOs with OAs
It has been reported that feed additives with different functions and complementary mode of actions hold the most promising solution to replace antibiotics in animal feed mainly for three reasons: (i) all the beneficial effects of antibiotics are unlikely to be covered by an individual alternative; (ii) some alternatives possess a synergetic effect that may decrease the required dose considered as cost-effective; (iii) substituting the antibiotics must be an integrated approach that includes feeding, management and biosecurity rather than a supplementation of feed additives alone [172].
As for the synergy between feed additives, the combined use of hydrophobic EOs with lipophilic OAs has been considered the most promising method to substitute antibiotics for the potential synergistic and additive beneficial effects on the intestinal health and growth performance compared with individual EOs or OAs [181,182]. Some results of recent studies are illustrated in Table 7. The main mode of action linked to the synergic effects of OAs and EOs may be the modulation of the intestinal microbiota. However, the antimicrobial activities depend on the gram staining of bacteria as Gram-negative (G−) bacteria differ from Gram-positive (G+) bacteria with the respect to the structure of the cell wall. The cell walls of G+ bacteria are 90-95% composed of peptidoglycan allowing hydrophobic molecules (EOs) to easily penetrate the cells, acting on both the cell walls and the cytoplasm, causing a disruption of the structure and function of bacteria cell membranes [183]. This increase of the bacterial membrane permeability could facilitate the influx of OAs into the cytoplasm due the lipophilic nature of their undissociated form, disturbing the proton and associated anion concentrations in the cytoplasm [182]. As for phenolic compounds, once inside the cell, they can interfere with enzymes involved in the production of energy at lower concentrations and denature proteins at higher concentration. However, G− bacteria possess a different composition as their peptidoglycan layer is only 2-3 mm thick and composes only 20% of the dry weight of the cell. An outer membrane comprised of a double layer of phospholipids firmly linked by Braun's lipoprotein to the inner membrane, lies outside of the peptidoglycan layer, making G− bacteria less permeable by providing an extra layer of protection and thus more resistant to EOs than the G+ bacteria [183]. Moreover, the "quid" provided by the core polysaccharides and the O-side chain allows these bacteria to be more resistant to EOs and other natural extracts possessing anti-microbial activities. Although the antimicrobial properties of long chain fatty acids (LCFA) are related to their potential to incorporate themselves into target membranes of G+ bacteria, promoting leakage of cellular protons and ions due to their lipophilic nature, the lipopolysaccharide (LPS) layer in the cell wall of G− bacteria prevents medium chain fatty acids (MCFA) and LCFA from crossing the cell membrane. Moreover, G− bacteria are also able to assimilate MCFA and LCFA into the cell and subsequently metabolize them per the β-oxidation cycle (i.e., Escherichia coli) and utilize short chain fatty acids (SCFA) as energy sources (i.e., Salmonella, Escherichia coli). These differences in the cell membrane compositions make EOs more powerful in the control of G+ bacteria compared to G− ones. However, OAs are reported to be more effective against G− bacteria than EOs [184] as small hydrophilic solutes of OAs are able to pass through the membrane via porin proteins but not the hydrophobic polyphenol molecules [172]. Table 7. Application of mixtures of phytogenics and organic acids with major physiological responses in poultry.  -Increased mRNA relative expression of aminopeptidase, sodium-glucose cotransporter 1, and Na+-independent neutral amino acid transporter in duodenum and glucose transporter 2 in jejunum with 300 mg/kg. -Higher mRNA relative expression ofmucin-2 in ileum with 300 mg/kg. -Linear decrease of the secretory immunoglobulin in ileum A. [188] The mechanism of inhibition to microorganisms by OAs may be affected by several factors, including the reduction in pH, the ratio of the un-disassociated form of the acid, chain length, degree of branching and cell physiology/metabolism [189]. Indeed, the lipophilic nature of weak organic acid allows them to easily penetrate the plasma membrane and thus acidify the cell's interior eventually killing the bacterium [172].

Conclusions
This review tried to gather the most recent available scientific information regarding the use of phytogenics in poultry nutrition along with their beneficial effects on performance, digestibility, microbiota, immune response, oxidant status, as well as egg and meat quality. Several studies have reported the promising effects of these feed additives when combined together or with organic acids; however, extra attention should be focused on the selection of active compounds to form potentially effective blends. Moreover, choosing the appropriate technique of protection as well as types and physiochemical properties of wall materials are the most critical aspects governing efficiency by controlling both the timing and location of the release of active compounds.

Conflicts of Interest:
The authors declare no conflict of interest.