Lycopene in Feed as Antioxidant and Immuno-Modulator Improves Broiler Chicken's Performance under Heat-Stress Conditions

Lycopene is a type of carotenoid pigment widely distributed in various plants and fruits, with tomatoes, carrots, and guava being the most abundant sources. Due to its high content of beneficial active components, lycopene has been used in medicine, where it is employed as a dietary additive for cancer therapy, immune modulator, and feed additive to improve livestock productivity. Lycopene is a lipophilic substance that can act as either a prooxidant or a free radical scavenger and is particularly efficient in enhancing broiler performance. Furthermore, lycopene can alleviate heat stress by improving the activity of various antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT), as well as increasing the total antioxidant capacity (T-AOC) and nuclear muscle factor erythroid 2-related factor 2 (Nrf2), while simultaneously reducing the levels of malondialdehyde (MDA) and muscle Keap1 expression. In addition, lycopene can improve broiler fertility by enhancing sperm performance and reducing inflammation by modulating the levels of interleukin 1, 2, and 10 (IL-1, IL-2, and IL-10) in cases of infection. In cases of disease by aflatoxin B1 (AFB1), lycopene can modulate interferon-γ (IFN-γ), IL-1, claudin-1 (CLDN-1), and zonula occludens-1 (ZO-1). Furthermore, under the lipopolysaccharide challenge, lycopene can increase the relative weights of immune organ indices such as the bursal, spleen, and thymus.


Introduction
Animal protein consumption rapidly increases annually following the development of the social economy and population growth. Poultry products mainly fulfl this demand. Furthermore, the industry is experiencing rapid growth, particularly in developing nations [1]. Lycopene (carotenoid group) is a natural antioxidant with promising properties as a feed supplement for broiler chickens, particularly under heat-stress conditions, a signifcant issue in global poultry farming [2,3]. Heat stress is correlated with the inability of animals to metabolize carotenoids, necessitating carotenoid supplementation through the diet [4]. Many fruits and vegetables provide natural carotenoids for broilers in limited amounts. Terefore, feeding these particular supplements to broilers seems appropriate for maintaining health and production.
Te concentration of lycopene in fresh tomatoes (Solanum pimpinellifolium L) ranges from 0.594 to 03.09 mg/ 100 g and can be infuenced by density, variety, and hue [5]. Commercial lycopene supplements are readily available although their content may vary depending on the type and formulation. Lycopene possesses notable antioxidant and prooxidant properties [6] and has been shown to mitigate the efects of reactive oxygen species (ROS) in broilers resulting from metabolic activities that generate free radicals [7]. Tese free radicals, unstable molecules composed of nitrogen and oxygen, can damage live cells. However, lycopene has demonstrated exceptional reactive oxygen species absorption capacity due to its conjugated structure generated from a double bond system.
Providing lycopene to broiler chickens exerts positive efects (Table 1), especially in crucial periods such as extreme temperature and infection-associated conditions [8]. Te inclusion of a commercial diet supplemented with lycopene has been shown in previous research to boost yolk pigmentation and immunity in chickens throughout production [22,23]. Terefore, this review explains the efects of lycopene in broiler chickens on their performance due to its antioxidant and immune-modulating properties.

Data Collection
Information about lycopene was gathered from various online databases such as PubMed, Google Scholar, Research Gate, Academia, and Elsevier. Te keywords in the data observation were lycopene in broiler chicken, lycopene as an antioxidant, and lycopene as an immune-modulators.

Source and Biochemistry of Lycopene
Lycopene can be obtained through chemical synthesis, fermentation, or extraction from various sources, including plants, algae, fungi, and microbes [24,25]. Natural sources of carotenoids, such as tomatoes, particularly the dark red variety and its byproducts, contain abundant lycopene. Other fruits, such as watermelon, guava, apricots, papaya, and pink grapefruit, also contain lycopene [26].
Te carotenoid family is divided into subgroups based on chemical or functional features. Carotenoids are categorized as primary or secondary based on their chemical structure [2,27]. Carotenoids are a group of isoprenoids with adaptive properties that play essential roles in plants and animals. Tese properties range from acting as cellular antioxidants to regulating gene expression. Terefore, their signifcance at the cellular and molecular levels is signifcant [28]. Berzelius extracted yellow pigments called xanthophylls from leaves in 1837. Tswett used chromatography to isolate and purify xanthophylls and carotenes and coined the term "carotenoids" in 1911. Between 1873 and 1927, Harsten and Willsta derived 11 g of lycopene from 75 kg of tomatoes, separating it from other carotenoids. Cámara et al. [5] later explained the chemical compositions of lycopene and other carotenoids, establishing correlations between the existence of conjugated double bonds, colour, and spectroscopic characteristics, as well as resemblances to the retinol molecule.
Lycopene belongs to the class of lipid-soluble carotenoids and is a typical isoprenoid molecule [29,30]. Te molecule contains several conjugated double bonds that give it its distinct red colour and make it easily detectable. It protects against oxidation or isomerization when exposed to oxygen, light, heat, acid, or other environmental stressors. It converts the native all-trans-isomers into cis-isomers and signifcantly impacts biosynthesis [31]. Due to its function as a carotenoid, lycopene can be utilized as a natural and lipidsoluble food colourant. According to previous studies, lycopene possesses anticancer, immunomodulatory, anticardiovascular, and antioxidant properties [29,32,33].

Dietary Intake Levels of Lycopene in Broiler Chicken
Poultry production is vulnerable to diseases and stress, such as pathogenic microbes and viruses, heat stress, extreme environmental conditions, and other imbalances that threaten broiler farming. According to the previous literature, a diet high in carotenoids boosted yolk pigmentation and immune protection against infection in broilers [2,3]. According to Koutsos et al. [37], the carotenoid level in the lymphoid organs, such as the thymus and bursa of Fabricius, was higher than that in the plasma and liver of broiler chickens with a weakened immune system. Te dietary supplementation with lycopene (12 mg/kg) and other compounds like fsh oil and selenium enhanced birds' productivity, nutrition rate, and redox status [38].

Antioxidant Sources and Effect of Lycopene in Heat-Stressed Broilers
As a dietary supplement, exogenous antioxidants are essential since they improve poultry productivity and meat quality and maintain health by boosting the immune system and metabolism [39]. Exogenous antioxidants protect the organism from oxidative damage induced by excess ROS as the byproduct of metabolism. Tese radicals negatively afect the organism if they undergo oxidative stress initiated by stressful conditions, including heat exposure [7]. Lycopene supplementation can reduce free radical levels, which may be attributable to its robust antioxidant properties connected to its more remarkable singlet oxygen quenching ability, double that of alphacarotene, and ten times that of alpha-tocopherol [40]. It eliminates free radicals by electron transfer, adduct formation, and hydrogen atom transfer [40]. Hence, lycopene simultaneously inhibits lipid peroxidation and DNA damage by inducing enzymes of the cellular antioxidant systems by triggering the transcription of antioxidant response elements [41]. According to Arain et al. [26], lycopene successfully increased levels of antioxidant enzymes (GSH-Px, SOD) and considerably decreased MDA content in muscle and blood. Moreover, adding 75 mg/kg lycopene to the broiler diet enhanced the oxidative stability of meat [9].

Animals
Similarly, Sahin et al. [20] indicated that increasing dietary lycopene concentration (20, 50, and 100 mg/kg food) linearly boosted serum activities of SOD and GSH-Px and lowered MDA concentration in heat-stressed broilers. Furthermore, Li et al. [42] reported that feeding broilers with tomato paste containing 10, 20, and 17 g/kg of lycopene resulted in a reduction of the levels of thiobarbituric acid-reactive substances (TBARS) and lowdensity lipoprotein (LDL) in broilers at weeks 2 and 4. Lycopene mediates the control of phase I and II detoxifying enzymes and gene transcription [43]. Te activation of the so-called antioxidant response element boosts the production of cellular enzymes, including quinone reductase (ARE), superoxide dismutase (SOD), and glutathione S-transferase (GST) [43,44]. A new study by Wang et al. [16] demonstrated that supplementing 1-day-old broilers with lycopene (particularly at 30 mg/kg) for 42 days signifcantly improved their blood antioxidant status, as evidenced by increased T-AOC, GSH-Px, and SOD activities and lowered MDA level on day 21 of the study. In addition, lycopene supplementation improved hepatic antioxidant capacity, increased GSH-Px and SOD activities, decreased MDA levels (10, 20, and 30 mg/kg lycopene), and increased GSH-Px activity (30 mg/ kg lycopene) in lever.
Additional research by Sahin [45] demonstrated that increasing lycopene intake from 200 to 400 mg/kg in Ross 308 broilers decreased muscle mass. Expression of Keap1 and Nrf2 in muscle was considerably more signifcant in the heat-stress (HS) environment than in the thermoneutral (TN) environment. In response to oxidative stress, the Nrf2 protein is a redox-sensitive transcription factor that stimulates the production of numerous enzymes to activate the phase II detoxifying/antioxidant system [46]. Increased expression of Keap1 and decreased expression of Nrf2 in the muscle of heat-stressed broilers may result from their activation and translocation in response to oxidative stress caused by heat-stress exposure [47]. Under oxidative stress, Nrf2 dissociates from Keap1, translocates to the nucleus, and forms a heterodimer with Maf protein to stimulate the transcription of downstream genes [16]. In an experiment conducted by Wan et al. [14], the addition of 100-400 mg/kg lycopene to the diet of broilers fed AFB1-contaminated diets for 42 days dramatically decreased the hepatic activities of cytochrome P450 2A6 (CYP2A6) and P450 1A1 (CYP1A1). Lycopene has exhibited resistance against oxidative damage induced by Afatoxin B1 (AFB1), as demonstrated by increased mRNA expression of the NRF2 signalling pathway and inhibition of Cyp450 isozymes [14,19].

Lycopene Increase Fertility of Birds
A study by Mangiagalli et al. [48] observed that lycopenesupplemented broilers had higher fertility rates than unsupplemented broiler breeders. In addition, the enhanced sperm progressive motility and membrane integrity caused by 0.2 mM lycopene-loaded nanoliposomes therapy likely increased the quantity of functional sperm in the sperm storage tubules, boosting fertility [13]. Moreover, boosting sperm cellular features by augmenting the antioxidant system and mitochondrial activity may improve sperm function during passage through the reproductive canal [49]. Tis improvement in fertility was not only due to a rise in the amount of surviving spermatozoa after freeze-thawing but also to a signifcant improvement in their functionality. As an antioxidant in reproduction organs, lycopene administration increased TAC and GPx while lowering MDA levels in sperm [13]. Tese variables are associated with enhanced sperm quality. However, Puzzo et al. [21] show that the administration of lycopene did not afect the lipid peroxidation of either breast or thigh meat. Likewise, lycopene supplementation did not afect the antioxidant response of liver and kidney tissues.

Immunomodulatory Effect of Lycopene in Broilers
Te efects of immunosuppressive agents on heat-stressed chickens have been demonstrated. Heat stress diminishes total antibodies and specifc IgG and IgM levels during primary and secondary humoral responses. In addition, it signifcantly lowers the weight and size of the liver, thymus, spleen, lymph, and bursa, followed by a decrease in the number of lymphocytes in the organs [50]. In chickens, however, a loss of lymphoid tissue has been observed. Moreover, several studies have reported that heat stress can infuence the number of circulating blood cells. Te ratio of heterophils to lymphocytes also increases due to heat stress [50][51][52]. Naturally, various bird species have utilized carotenoids from fruits or leaves as potent immune-modulating antioxidants to combat cytotoxic reactive oxygen species [53] due to heat stress, which can increase susceptibility to infammation [10]. Moreover, it induces epithelial barrier destruction, and immune cell migration contributes to the augmentation of tissue infammation in some organs caused by NF-B as the transcription factor that plays an essential role in most chronic diseases' development, maintenance, and progression [45,54].
As the most substantial antioxidant component in carotenoid groups, lycopene has a potential antiinfammatory efect [42,55,56]. Terefore, it can ameliorate intestinal injuries and increase the epithelial integrity of the digestive tract [19]. Sarker et al. [15] showed that lycopene inhibited INF-c and upregulated IL-10 in the intestinal epithelium of afatoxin B1-infected broilers. It is widely accepted that IL-10 is a strong antiinfammatory cytokine that suppresses the proinfammatory cytokines generation and dampens immune responses [57]. Moreover, lycopene lowered adhesion molecules, infammatory genes, and proinfammatory cytokines [58]. Tese alterations may be attributed to the fact that cytokines are critical components of the immune system and play an essential role in defending against pathogenic bacteria [59].
Several studies have shown that lycopene supplementation can improve the immune response in broiler chickens and other animal species. For example, lycopene treatment has been reported to reduce CRP, haemoglobin, and serum TC levels in broilers infected with E. coli while also increasing IL-10 and phagocytic activity and decreasing IL-1 [40]. Similarly, Yonar [60] found that lycopene treatment increased oxytetracycline-suppressed phagocytic activity in rainbow trout. Lycopene's immunostimulant action may explain its ability to increase immunological parameters by boosting the synthesis of interleukin-2 (IL-2) and interferon-gamma-c (INF-c), which are potent activators of T cells [61]. Furthermore, lycopene may encourage the immune system to combat oxidative damage to lymphocytes' DNA [62].
Dietary lycopene at 200 mg/kg could alleviate intestinal damages caused by AFB1 by diminishing infammatory responses and decreasing oxidative stress of the intestinal mucosa, as suggested by increased IL-the interferon-c (IFNc) levels (IL-1) and increasing mRNA abundances of Cludin-1 (CLDN-1) and (ZO-1) in the jejunum [19]. CLDN1 and ZO-1 are tight junctions' most important structural and functional components and defend against pathogen growth [63]. Also, in an experiment by Wan et al. [14], the addition of 100-400 mg/kg lycopene to the feed of broilers fed AFB1-contaminated diets for 42 days decreased the blood levels of alanine aminotransferase (ALT) and aspartate transaminase (AST).
Furthermore, feeding broilers with diets supplemented at 100-400 mg/kg of lycopene lowered the activity of ACC, REBP-1, and FAS and increased the mRNA expression level of AMPK [16]. Hence, lycopene-supplemented diets in broilers could stimulate the AMPK pathway system, thereby reducing fat synthesis in the liver. Activating the AMPK pathway system may account for decreased abdominal fat formation and controlled serum lipid levels. Sun et al. [64] investigated the benefcial efects of lycopene on breeding hens exposed to lipopolysaccharide and found that feeding hen's lycopene (20-80 mg/kg) for 35 days enhanced the immune organ indices of the bursal, spleen, and thymus. However, a sizeable supplementary dose of lycopene may induce adverse efects in livestock. According to Pozzo et al. [21], supplementing the diets of Hubbard broilers with up to 500 mg/kg of lycopene lowered the weight of the bursa of Fabricius and spleen.
In addition, the increase in white blood cells, notably the proportion of lymphoid cells, and the decrease in the fraction of diferentiated lymphoid cells may be attributable to the antioxidant activity of lycopene [65]. However, Pozzo et al. [21] reported that 35 days of feeding Hubbard chickens 500 mg/kg of lycopene decreased total protein, alpha globulin, gamma globulin, and albumin. In addition, an increase in apoptotic cells was identifed in the spleen and bursa of Fabricius samples from lycopene-fed hens [21].

Conclusion
Lycopene supplementation can reduce free radical levels and protect the organism from oxidative damage induced by excess ROS due to heat exposure. Lycopene simultaneously inhibits lipid peroxidation and DNA damage by inducing enzymes of the cellular antioxidant defence systems by triggering the transcription of antioxidant response elements. Dietary lycopene in the feed also can increase the serum antioxidant state, lower MDA content, and boost the antioxidant enzyme activity in the blood of heat-stressed broilers. In addition, lycopene mediates the control of phase I and phase II detoxifying enzymes and gene transcription and increases fertility rates in broiler breeders.

Data Availability
A search of electronic databases including PubMed, Elsevier, Research Gate, Academia, and Google Scholar yielded information about lycopene in broiler chicken. Tese search terms were utilized: lycopene, broiler performance, broiler production, antioxidants, and immunomodulators. In the article, the research data are presented in table format. Previous studies cited in a recent journal that is relevant to the topic of this article provided data for discussion and comparison purposes. Tese data are publicly accessible via the Internet. Tere are extensive citations in the manuscript's bibliography.

Conflicts of Interest
Te authors declare that there are no conficts of interest in this study.