1. Introduction
The poultry gastrointestinal tract (GIT) offers a biological environment for nutrient digestion and absorption, as well as protection from pathogens and toxins. Oxidative stress in birds’ GIT is derived from nutritional factors, environmental heat stress, and pathological factors, which alter the overall performance as well as meat quality [
1,
2]. Biological damage associated with oxidative stress can cause many degenerative health issues, which have a great impact on the overall performance and productivity of livestock [
3]. Studies have suggested that the interaction of mucosa with microbes or their toxins triggers oxidative stress [
4,
5,
6]. The supplementation of antioxidant-rich diets and plant extracts having antioxidant properties that scavenge reactive oxygen species (ROS) are beneficial in mitigating oxidative stress in the GITs of animals [
7,
8,
9]. Avian coccidiosis is among the most common parasitic diseases caused by genus
Eimeria spp. in the poultry industry that is responsible for great economic losses, increasing mortality and lowering growth rates [
10].
Eimeria spp., primarily producing proinflammatory mediators together with oxidative stress, contributes to lipid peroxidation, antioxidant insults, damage of the intestinal epithelial barrier, inflammatory injury and diarrhea [
11]. Following infection with parasites, particularly with
Eimeria spp., the antioxidant systems of chickens are significantly disrupted. The use of antioxidants is critical in combating the oxidative stressors caused by the production of ROS and for the maintenance of homeostasis [
12]. Under the conditions of ROS overproduction, supplementation with compounds of high antioxidant potential is immensely valuable [
13,
14]. Concomitantly, regarding the prolonged use of common traditional approaches, such as coccidacidal pharmaceuticals that are used for controlling avian coccidiosis, these chemotherapeutics have been banned due to decreased
Eimeria spp. sensitivity and developed drug resistance [
15,
16]. Therefore, the dietary inclusion of bioactive compounds that have new antioxidant and immunological prophylactic properties can solve these previous issues by exerting specific coccidiastat effects [
17,
18]. Prior studies have recommended that natural phytogenic-derived agents, such as resveratrol, oregano essential oil and aloe vera, have profound impacts on ameliorating oxidative stress for animals [
19,
20]. Moreover, phytogenic substances, rich in isoflavones, have been recognized as alternative additives to replace antibiotic usage in poultry farming via enhancing intestinal integrity and controlling inflammatory signaling pathways [
21]. The anticoccidial properties of several natural herbal products (or their extracts) is mainly attributed to their ability to lower the impact on the output of oocyst via the inhibition or suppression of the invasion, replication and expansion of
Eimeria ssp. in chickens’ gastrointestinal tissues [
22].
Moreover, the therapeutic potential and defensive role of these phytogenics against artificially induced coccidiosis in chickens is mainly due to their phenolic compounds that interact with cytoplasmic membranes, triggering coccidial sporozoite death, attenuating intestinal lipid peroxidation, facilitating epithelial injury repair and lessening the intestinal permeability triggered by
Eimeria spp. [
23]. Phenolic compounds, such as flavones (apigenin and luteolin), hydroxycinnamic, caffeic and sinapic acids, have been tested for their inhibitory effective role on the sporulation of coccidian oocysts [
24,
25,
26]. Thus, searching for a new bioactive phenolics candidate with anticoccidial efficacy is an important prerequisite. Myricetin is a natural flavonoid with known strong antioxidant, anti-inflammatory and anticancer properties [
27,
28]. Additionally, the antiparasitic effect of myricetin was recently documented against Schistosomiasis [
29]. The potential antioxidant activity of myricetin could be related to the presence of three hydroxyl groups on its B ring like other flavonoids [
30]. Furthermore, myricetin has also proved to modify inflammatory diseases by suppressing pro-inflammatory mediators (inhibition of inflammatory mediators such as TNF-α, IL-6, IL-12 and iNOS) [
31,
32]. Myricetin can effectively reduce oxidative stress via down-stream expression of HO-1 and NQ1 [
33]. At present, the impact of myricetin on combating oxidative stress and avian coccidiosis has not been reported yet in the poultry industry. Hence, this study was designed to assess the effective role of in-feed myricetin on broiler chickens’ performance indices, antioxidant and immune modulation, fecal oocyst excretion and intestinal lesion score following
Eimeria ssp. challenges.
4. Discussion
The distribution of the oxidative balance that emerges from oxidative stress, owing to higher free radical production, can trigger cell chain reactions, which in turn results in cell damage or even death. Invasion by the
Eimeria species can evoke the over production of ROS and free radicals in the host’s cellular immune response and goes beyond the protection capability of the natural antioxidant defense system, which contributes to tissue damage and pathological lesions [
45]. Oxidative stress can trigger inflammation that modifies gene expression related to antioxidant status, playing a critical role in the physiological function of the gastro intestinal tract [
46]. Antioxidant-enriched diets are among the most substantial dietary factors for poultry, which have unique consequences not only for maintaining their better growth and preventing them from various diseases but also affecting the quality of their products offered for consumers. Plants or their bioactive principles enriched with flavonoids are considered an alternative approach to treat coccidiosis [
47]. Herein, dietary myricetin harbors strong antioxidants properties that attenuate the impaired host oxidative equilibrium resulting from coccidian experimental infection. During the starter stage, the dietary inclusion of myricetin in broilers’ diets improved body weight gain and the efficiency of the feed conversion ratio. In accordance, feeding with dietary polyphenol-enriched grape seeds improves the body weight gain and antioxidant status of broiler chicks [
48]. In many studies, plant-derived flavonoids have been reported to have an effective role in reducing lipid oxidation, decreasing the pathogenic microbial loads in birds’ intestines and intestinal pH and improving the histomorphology of the intestine, leading to maximizing nutrient absorption and promoting growth performance [
49,
50]. Myricetin is a plant-derived flavonoid that exhibits many activities in animals’ bodies as a growth promoter with anti-inflammatory, antioxidant and anticancer properties [
51]. Before infection, dietary myricetin enhances the immune response and antioxidant status of birds and consequently enhances their performance, including improving their growth rate and FCR. Coccidiosis can induce intestinal oxidative stress that greatly impairs the growth rate and feed efficiency of birds [
11]. Remarkably, after
Eimeria spp. challenges, the body gain of birds was greatly impaired in the IC group that was fed with no additives, and the Myc-supplemented groups, especially at 600 mg/kg, restored these impaired growth performance parameters. Similar results were reported by Bozkurt et al. [
52], as the growth performance of broilers and feed conversion ratio worsened after coccidial infection. Moreover, the main clinical signs of coccidial infection are reductions in body weight gain and feed intake with high feed conversion and high economic losses. The alleviated growth performance due to flavonoid supplementation is in agreement with the findings of Wang et al. [
47], who reported improved growth performance of experimentally coccidial-infected birds fed with diets supplemented by grape seed extract. The improvement of the birds’ performance may be attributed to the anti-inflammatory effect of flavonoids that reduces the effect of the coccidial destructive effect on the intestine, ameliorating intestinal health status and decreasing diarrhea [
53]. Moreover, the anticipated mechanism of action of flavonoids also resulted from their hydroxyl groups, which acted as pro-oxidants that oxidized via ROS inside cell membranes, in turn delaying the bad consequences (lipid oxidation and DNA damage) [
54].
In the current study, we assessed the efficacy of myricetin against the severity of coccidian infection via evaluating fecal oocyst counts after infection at 7, 14 and 21 dpi. Coccidian infection exaggerated oocyst excretion per gram of feces after 7 dpi, especially in the infected non supplemented control. The rate of oocyst excretion in feces was observed to decrease as the number of days post infection increased in all groups. Interestingly, the rate of oocyst excretion was much lower in groups supplemented with higher doses of dietary myricetin compared to that of the infected control. In correlation, the intestinal lesion score in all segments of the intestine was reduced with increasing the supplementation levels of myricetin. Phytochemicals as herbal plant extracts or their active substances with antioxidant functions have been developed to be used as anti-parasitic agents, especially coccidiosis such as garlic extract [
55], cinnamaldhyde [
56], Chinese herbs [
57] and a mixture of thyme, oregano and garlic [
58]. The effective role of flavonoids and polyphenols in reducing oocyst shedding in the feces of broilers was in accordance with the findings of [
16,
59], in which it was found that the addition of herbal extract enriched with flavonoids reduces coccidial oocyst shedding in feces simultaneously with decreasing the severity of infection. Moreover, our findings are in accordance with Wang et al. [
47], who observed decreased fecal oocysts and intestinal lesion scores with increased levels of grape seed extract in broiler chickens. In line with our findings, Liu et al. [
53] reported that chlorogenic acid, which is an antioxidant and anti-inflammatory substance that reduces oocyst count in feces, intestinal lesion score and bloody diarrhea, indicates the inhibition of coccidial infection in broiler chickens. The coccidiocidal or coccidiostatic role of flavonoids could be attributed to interrupting the parasitic life cycle via inhibiting its sporulation [
60]. Additionally, the main role of flavonoids and polyphenols as anti-coccidials may be due to its mode of action as anti-inflammatory and antioxidant substances that improve gut health through maintaining mucus secretion, increasing gut epithelial integrity, reducing the colonization of pathogenic microbes and improving local intestinal and body immune defense [
61,
62].
Dietary polyphenolic supplementation can improve the immune systems of the birds via several ways: binding to the immune cells’ receptors and changing the signaling pathway of the cell, causing the regulation and modulation of the immune response of the host against invasive microorganisms; and enhancing the release of anti-inflammatory cytokines that improve the birds’ resistance against infection [
63]. The enhanced immunity that decreased fecal oocyst shedding, intestinal lesions and bloody diarrhea due to myricetin supplementation, from our point of view, is the main cause of the decreased mortality percent compared to the IC group. In accordance, reduction in mortality due to coccidial infection was also observed in broiler chickens supplemented with grape seed extract, which is rich in flavonoids and polyphenol compounds [
47]. Moreover, Ageratum conyzoides, enriched with flavonoids at the level of 500–1000 mg/kg, revealed a considerable decrease in the oxidative stress produced by
E. tenella, improving broiler chickens’ performance and reducing mortality [
64].
Under normal management conditions of broiler chickens, the dietary supplementation of polyphenols and flavonoids has a crucial role in protecting birds from oxidative stressors and neutralizing the free radicals produced in body cells such as ROS and reactive nitrogen species (RNS) [
65]. Plant-derived polyphenolics compounds have been proven to play an important role in the stimulation of immunity, either through cellular immunity through the modulation of the function of immune cells by binding to immune cells receptors, altering their signaling pathway and stimulating their proliferation [
66]; or humoral immunity through the elevation of an antibody titer, increasing lysozyme activity and increasing serum immune globulins [
67]. Herein, the role of myricetin (400 and 600 mg/kg) in stimulating the immune status of broiler chickens before infection was clear through boosting IgG and lowering CRP levels.
Parasitic infection such as by
Eimeria spp. can induce inflammatory responses of the host [
53]. In our results, after coccidial challenge on day 14, the NO, CRP and MPO levels were significantly elevated in the IC group compared to the non-infected one, and myricetin addition significantly decreased their levels.
Eimeria spp. infection has been reported to induce plasma NO levels that may be involved in their pathogenesis, as follows; NO is considered to be a toxic substance to sporulated oocysts [
68], and the ingestion of NOS inhibitors increases oocyst output [
69]. Also, Yan et al. [
68] proved that exogenous NO causes the egress of
E. tenella sporozite from primary chicken kidney cell cultures before parasite replication. However, the high production of NO by the host cell above the host cell’s tolerance due to coccidian infection can cause tissue damage and cell cytotoxicity, which can induce the inflammation and development of clinical signs such as diarrhea, mortality and intestinal lesions [
70]. C-reactive protein, considered an acute inflammatory protein that lowers inflammation levels and is highly produced at the site of inflammation or infection by many cells, such as macrophages, lymphocytes and endothelial cells, is considered potential marker of decreased body inflammation and cells damage [
71]. Moreover, CRP plays a crucial role in response to the host’s infection through NO release, phagocytosis, apoptosis and cytokine production, particularly IL-6 and TNF-α [
72]. Myeloperoxidase is a pro-inflammatory enzyme generated from neutrophilic granulocytes, and it plays an important role in innate cellular immune responses through its potential effect to injure healthy tissue, thus contributing to disease initiation in poultry [
73]. Flavonoid supplementation, such as with curcumin, resveratrol and thymol, have been proven to have an immunostimulatory effect through inhibiting the generation of ROS and NOS by supressing MPO and reducing MPO mRNA expression in neutrophiles [
74,
75]. Additionally, the hummoral immune response of the host has been reported to be activated through an increased antibody titer, espcially the protective IgG titer, after coccidial infection in laying hens [
76]. Our results indicate a high titer of IgG in infected birds supplemented with myricetin, escially at higher dosese. Similarly, Liu et al. [
53], who reported an improved antibody titer in
Eimeria spp. challenged birds, fed birds a diet supplemented with antioxidant chlorogenic acid. Notably, at 21 dpi, the excessive inflammatory response in the IC group subsided in myricetin-supplemented groups, which indicates its potent role against coccidial challenges.
The protective consequence of dietary myricetin against coccidian infection in our study was also achieved through the downregulation of proinflammatory cytokines (
IL-1β,
IL-6 and
TNF-α) and chemokines (
CCL4,
CCL20 and
CXCL13) and the upregulation of anti-inflammatory cytokines (
IL-10) and
AvBD6 and
AvBD612 mRNA gene expression. In agreement with our observations, flavonoids such as resveratrol have been proven to have a role in the generation and modulation of cytokines and chemokines in different immune cells [
77]. The essential role of proinflammatory cytokines such as interleukin 1 (IL-1), IL-6 and TNF-α have a responsibility in the acute-phase inflammation that is associated with general and metabolic changes [
78]. Moreover, these proinflammatory cytokines have a crucial role in modulating the host immune response during infection [
79]. Moreover, the anti-inflammatory cytokine IL-10 has been reported to have a role in controlling the host’s immune response by limiting the target cell damage during inflammation [
80]. Parasite invasion can use IL-10 to downregulate host immunity and reduce pathogen-damaging inflammatory reactions [
81].
Macrophage inflammatory proteins are also known as chemotactic cytokines that comprise CCL4 and CCL20, which play an important role in coordinating the host’s immune responses against infection [
82]. Furthermore, CCL4 acts as chemoattractant for important immune cells such as monocytes, macrophages, T-lymphocytes, dendritic cells and natural killer cells [
83]. Moreover, CCL4 secretion from neutrophils participates in inflammation by attracting other leukocytic cells to the area of inflammation, resulting in resolving the inflammation by macrophage-mediated cells and developing chronic inflammation [
84]. Regarding the CCL20 chemokine, it plays a vital role in the initiation of chronic intestinal inflammation in broiler chickens [
85]. CXCL13, which is also recognized as stromal cell-derived factor-1, has a high chemotactic impact on lymphocytes that are involved in inflammatory responses of the host against infections [
86]. Moreover, resveratrol was found to improve phagocytes’ killing capability, and the inhibition of TNF-α and NF-kB was found to relieve inflammation in damaged livers with hydrogen peroxide [
87]. In the same vein, the immune responses of tilapia have been reported to be improved by the upregulation of
IL-10 and
TGF-β and the downregulation of
IL-1β,
IL-8 and
TNF-α mRNA levels after supplementation with quercetin nano particles [
88].
Defensin is an indispensable peptide for the host’s defense mechanism, giving it instant defense against microbial invasion. However, the particular role of defensin proteins in local resistance against the infection of
Eimeria ssp. has not been well explored [
89]. Avian β-defensin 6 and 12 exhibit a chemotactic effect and lipopolysaccharide-neutralizing effect for chicken macrophages. In addition, AvBD12 has been proven to be involved in the induction of murine immature dendritic cell migration to the site of inflammation [
90]. Herein, increasing the expression of AvBD6 and 12, following dietary supplementation with Myc, indicates its protective role against coccidia infection.
Additionally, COX-2 is an enzyme whose intermediate, arachidonic acid, undergoes bioconversion to inflammatory prostaglandin with consequential cytokine release [
91].
Invasion of host cells with
Eimeria spp. is known to produce oxidative stress through releasing high amounts of free radicals that play a crucial role in the host’s defense mechanism against parasite infection [
92]. The concentration of those free radicals may increase cell tolerance and cause cell cytotoxicity and death, cascading the pathogenesis of the disease. Moreover, the production of this massive amount of ROS and NOS in parasitic diseases can exhaust both low molecular antioxidants such as vitamin A, E and C [
70] and metal-dependent antioxidants such as GPX, SOD and CAT [
93]. In the current study, it seemed that, after coccidia invasion, higher free radical release and high levels of NO and MDA production were the most important factors impairing the natural antioxidant defense system, which comes in agreement with Georgieva, Koinarski [
14].
As evidenced in our study of post coccidia challenge, ROS production decreased, and the expression of the
COX-2 gene in the group that was fed with higher Myc levels was downregulated, which are the main messengers that modify the expression of numerous genes implicated in inflammation [
94]. Furthermore, coccidia infection greatly downregulated the expression of antioxidant-related genes such as
GPX,
SOD,
CAT,
HO-1 and
NQO1 in both intestinal and muscle tissues, and the supplementation of strong flavanol compounds such as myricetin alleviated oxidative stress and improved the expression of these antioxidants. Herein, the contents of MDA and ROS and H
2O
2 levels in intestinal and breast muscle tissues were significantly reduced after the inclusion of elevated levels of Myc. In contrast, higher T-AOC in intestinal and muscle tissues following supplementation with Myc indicated decreased free radical production and lipid peroxidation. These findings suggest that feeding with myricetin strengthens the oxidative stability of birds via activating antioxidant mediators. The role of plant extracts in protection against coccidiosis may be related to their ability to control the impact on lipid peroxidation in intestinal mucosa and decreasing ROS and NOS production and consequently their destructive effects [
95]. The same results were obtained by Idris et al. [
96], who described that the inclusion of antioxidant-enriched essential oils can alleviate the oxidative stress caused by the invasion of
Eimeria spp. In agreement with our results, Tsiouris et al. [
16] reported improved antioxidant markers and the alleviation of coccidian infection occurring after the addition of high-polyphenol herbal extract.