Avian Cell Culture Models to Study Immunomodulatory Properties of Bioactive Products

Simple Summary Bioactive products have an effect on the molecular and biochemical functions of a living organism, causing a physiological response of the given tissue. Such a products are biologically active. Depending on the active component and amount, the effects of such products can be positive or negative. Bioactive products can be food ingredients or dietary supplements, and while they are not required for survival, they are responsible for changes in the body’s health. Poultry farming struggles with zoonoses and other infectious diseases that require the use of veterinary drugs such as antibiotics. However, it is preferable to increase the natural potential of the poultry to cope with the burden of innate immune responses. Bioactive products can be used as an alternative to microbial or antiparasitic agents. Over 400,000 different plant species contain bioactive chemicals, yet only a portion of them have been examined. To examine and describe their therapeutic capabilities, more scientific analyses and characterizations are required. The use of in vitro and ex vivo models enables the evaluation of the immunomodulatory effect of bioactive molecules derived from substances such as plant extracts, essential oils, probiotics, prebiotics, and synbiotics. This article presents several studies on bioactive products and their immunomodulatory effects tested in vitro and ex vivo using various avian models. Abstract Antimicrobial resistance is becoming a greater danger to both human and animal health, reducing the capacity to treat bacterial infections and increasing the risk of morbidity and mortality from resistant bacteria. Antimicrobial efficacy in the treatment of bacterial infections is still a major concern in both veterinary and human medicine. Antimicrobials can be replaced with bioactive products. Only a small number of plant species have been studied in respect to their bioactive compounds. More research is needed to characterize and evaluate the therapeutic properties of the plant extracts. Due to the more and more common phenomenon of antimicrobial resistance, poultry farming requires the use of natural alternatives to veterinary antibiotics that have an immunomodulatory effect. These include a variety of bioactive products, such as plant extracts, essential oils, probiotics, prebiotics, and synbiotics. This article presents several studies on bioactive products and their immunomodulatory effects tested in vitro and ex vivo using various avian cell culture models. Primary cell cultures that have been established to study the immune response in chickens include peripheral blood mononuclear cells (PBMCs), intestinal epithelial cells (IEC), and bone marrow-derived dendritic cells (BMDCs). Chicken lymphatic lines that can be used to study immune responses are mainly: chicken B cells infected with avian leukemia RAV-1 virus (DT40), macrophage-like cell line (HD11), and a spleen-derived macrophage cell line (MQ-NCSU). Ex vivo organ cultures combine in vitro and in vivo studies, as this model is based on fragments of organs or tissues grown in vitro. As such, it mimics the natural reactions of organisms, but under controlled conditions. Most ex vivo organ cultures of chickens are derived from the ileum and are used to model the interaction between the gastrointestinal tract and the microbiota. In conclusion, the use of in vitro and ex vivo models allows for numerous experimental replications in a short period, with little or no ethical constraints and limited confounding factors.

Abstract: Antimicrobial resistance is becoming a greater danger to both human and animal health, reducing the capacity to treat bacterial infections and increasing the risk of morbidity and mortality from resistant bacteria. Antimicrobial efficacy in the treatment of bacterial infections is still a major concern in both veterinary and human medicine. Antimicrobials can be replaced with bioactive products. Only a small number of plant species have been studied in respect to their bioactive compounds. More research is needed to characterize and evaluate the therapeutic properties of the plant extracts. Due to the more and more common phenomenon of antimicrobial resistance, poultry farming requires the use of natural alternatives to veterinary antibiotics that have an immunomodulatory effect. These include a variety of bioactive products, such as plant extracts, essential oils, probiotics, prebiotics, and synbiotics. This article presents several studies on bioactive products and their immunomodulatory effects tested in vitro and ex vivo using various avian cell culture models. Primary cell cultures that have been established to study the immune response in chickens include peripheral blood mononuclear cells (PBMCs), intestinal epithelial cells (IEC), and bone marrow-derived dendritic cells (BMDCs). Chicken lymphatic lines that can be used to study immune responses are mainly: chicken B cells infected with avian leukemia RAV-1 virus (DT40), macrophage-like cell line (HD11), and a spleen-derived macrophage cell line (MQ-NCSU). Ex vivo organ cultures combine in vitro and in vivo studies, as this model is based on fragments of organs or tissues grown in vitro. As such, it mimics the natural reactions of organisms, but under controlled conditions. Most ex vivo organ cultures of chickens are derived from the ileum and are used to model the interaction between the gastrointestinal tract and the microbiota. In conclusion, the use of in vitro and ex vivo models allows for numerous experimental replications in a short period, with little or no ethical constraints and limited confounding factors.

Introduction
Poultry production in Poland develops dynamically, especially in the broiler sector. Due to the large scale of the poultry production, the use of antibiotics is often required to reduce disease outbreaks [1]. Since 2009, the European Union has implemented legislation that aims to reduce the number of veterinary antibiotics in the poultry production. The use of antibiotic growth promoters in the chicken industry has been also reduced due to rising bacterial resistance to synthetic antibiotics and greater public awareness of health and food safety issues. This issue prompted researchers, the poultry industry, and the sector at large to look for safe alternatives to antibiotic growth promoters and focus on creating better long-term feed management solutions to boost chicken intestinal health and growth [2]. The trend to reduce antibiotics is correlated with the increased use of bioactive products that can have a positive effect on poultry growth, immune system, and health [3]. It is also worth noting that better animal health may lead to better food safety and quality, which benefits the consumers [4].
Bioactive products have an effort on the molecular and biochemical functions of living organisms, causing the physiological response of a given tissue. Such products are biologically active. Depending on the active component and dosage, the effects of such products can be positive or negative. Bioactive products consist of molecules that can have therapeutic effects such as reducing proinflammatory states, oxidative stress, and metabolic disorders [5]. Bioactive products can be food ingredients or dietary supplements, and while they are not required for survival, they are responsible for changes in the body's health [6]. Sources of bioactive products are plants (herbs and spices) and certain foods (fruits, vegetables, nuts, oils, and whole grains), but are also found in living organisms and microorganisms such as bacteria or fungi [6].
Immunomodulation refers to any process that modifies the immune system or immune responses triggered by an immunomodulator [7]. Immune responses can be enhanced with immunostimulants or natural compounds, synthetic chemicals, and microorganisms that are capable of modulating the function of the immune system [8]. Many immunomodulators are synthetic or semi-synthetic, and there is growing interest in natural compounds. Consumers consider natural products with pharmacological properties and therapeutic effects as being safer than manufactured chemicals. Natural compounds originating from plants and microbes have long been recognized as useful in drug discovery and development [9].
There is a close interaction between a balanced diet and healthy guts [10]. Many studies confirm that the condition of the intestinal microbiota is directly related to the development and maintenance of the immune system in animals [11]. Early feeding with a properly selected bioactive product causes colonization of the intestines with beneficial microbiota. The interaction of microbiota with the host has positive effects on the maturation of the immune system of chicks, and induces sufficient immune responses in growing chickens [12]. The basic role of the intestines is to process food through digestion and absorption of various nutrients [13]. The supply of the nutrients to the milieu of the body is also associated with the metabolic activity of commensal or pathogenic microorganisms colonizing the intestines. The intestinal immune system must be constantly ready to react and fight pathogenic microbes, while at the same time it must be tolerant towards the commensal microorganisms inhabiting the intestines [14]. Gut-associated lymphoid tissue (GALT) comprises the intestinal part of the mucosa-associated lymphoid tissue (MALT). GALT in chickens consists of lymphoid cells and follicles, Meckel's diverticulum, Peyer's patches, ceacal tonsils, and bursa of Fabricius. These structures include enterocytes, goblet cells responsible for the production of the mucus, cells that produce hormones, and peptides with antimicrobial activity. Both innate and adaptive immune responses take part in GALT. The immune response is initiated with antigens sensed by respective receptors.
Recognition of the antigen by the receptor triggers cellular signal transduction cascade, followed by cytokine secretion (cellular immune response), e.g., interleukin 10 (IL-10) or the transforming growth factor β (TGF-β). The goal of immune responses exerted by GALT is to neutralize pathogenic microbes while not affecting commensal organisms [15]. GALT is also the source of the largest population of B and T lymphocytes, which mediate specific immune responses and immune memory. Triggered by the contact with the antigen, B and T lymphocytes migrate to effector sites, where they develop specific immune responses of the intestinal mucosa.
Chicken is a well-established model for research on immune system and immune mechanisms. Using a chicken cell culture system as a model to study the immunomodulatory effects of bioactive molecules is more beneficial than using a live animal. The in vitro model is widely used due to the wide selection of options-primary cell cultures, cell lines, and ex vivo organ cultures including enteroids and organoids. It also allows for more repetitions of an experiment in a shorter time compared to in vivo models. In vitro models can also be used as a preliminary step for in vivo testing, which saves laboratory animals that otherwise would have to be sacrificed. Due to anatomical or physiological differences, it is very important to use species-specific cells or tissue cultures. This approach resulted in the intensive and rapid development of new in vitro models. The primary differences between avian and mammalian immune systems is the presence of the bursa of Fabricius, which in birds is a site of B cell development and the absence of encapsulated lymph nodes, which are replaced by the diffused lymphoid tissue in birds [10,11,[16][17][18]. Data obtained with cell culture experiments translate better to in vivo studies when the in vitro research model reflects the complexity of the tissue. This is particularly true for the immune response, which engages a wide repertoire of lymphoid cells.
In this article, we review selected bioactive products and their immunomodulatory effects in poultry. We also present an overview of the avian cell culture models that facilitate the study of immunomodulatory effects in vitro and ex vivo. Due to the wide range of possibilities regarding both the compounds used and cell culture research models, it is worth becoming acquainted with the state of knowledge on this topic. Immunostimulation is important because healthy intestines are responsible for the better absorption of ingredients, and thus the better growth and health of individuals.

Plant Extracts
Plant extracts, such as thyme, oregano, or cinnamon, are widely used in poultry production [3,19]. Plant extracts supplemented in animal feed contain bioactive compounds that improve appetite, digestion, and prevent certain pathological conditions [20]. Bioactive compounds are plant chemicals, so-called phytochemicals, that have a positive effect on the various physiological functions of their consumer, including immune responses and health [21][22][23][24]. The influence of the phytogenic feed additives, such as herbs, spices, essential oils, or various mixtures on the health traits of different animal species is well documented. These products have been used as natural growth promoters in pigs and poultry [19,20,22,25]. Plant extracts (oregano, laurel, sage, anise, and citrus essential oils) have a positive effect on slaughter performance and the health of broiler chickens [26,27]. Some herbs and spices, including turmeric, cumin, black, and red peppers, nutmeg, mint, ginger, as well as chamomile or anise, also have immunostimulating properties [19]. The mechanisms of action of many phytochemicals have been established using avian cell lines and cultured lymphocytes [28]. Cinnamon has been tested for its immunomodulatory properties and was found to have antibacterial, antioxidant, and anti-cancer properties. Lee et al. (2011) stimulated chicken spleen lymphocytes with 25 mg/mL cinnamaldehyde (a component of cinnamon) and found increased cell proliferation when compared to the control [29]. Lower concentrations of cinamoldehydeneotheli caused the activation of macrophage growth or the inhibition of tumor cell growth [29]. Based on studies performed on in vitro and in vivo models, it is believed that plants such as rosemary or thyme may serve as alternatives to coccidiostatics [30]. Such testing is typically performed using Eimeria spp. challenged with different bioactive products, which directly indicates the coccidias' sensitivity to a given compound. Studies have shown that the addition of plant extract from Bidens pilosa inhibited the penetration of parasites into intestinal cells in chickens [31]. Numerous studies show the ability of phytochemicals to prevent diseases or strengthen the immunity of chickens, but there is still a lack of information about the underlying mechanisms [8,14,16,22,23,32,33].

Prebiotics, Probiotics, and Synbiotics
Many bioactive compounds have immunomodulatory properties, and can affect the intestinal microbiota, as well as the host's immune system. Some of them are described below. Prebiotics, probiotics, and synbiotics affect the development of a healthy intestinal microbiota and inhibit the development of intestinal pathogens [34].
Prebiotics are oligosaccharides that are not fermented by the host, but only by the intestinal microbiota. This way, they support the growth of the intestinal microbiota [35]. Commonly used prebiotics in poultry include inulin, fructooligosaccharides (FOS), galactooligosaccharides (GOS), soybeans-oligosaccharides (SOS), xylo-oligosaccharides (XOS), pyrodektrins, isomacaccharides (IMO), and lactulose [36]. Prebiotics are tested in vivo on livestock to determine their effects on gut microbiota, host immunomodulation, and the inhibition of microbial infections [36]. Bednarczyk et al. (2016) used three different prebiotics administered in different ways (injected in ovo, in water, or combined methods) to stimulate the intestinal microflora populations of broiler chickens. All of the methods and the three prebiotics increased feed intake and the feed conversion ratio compared to the control. Such results suggest a balanced consumption of energy and nutrients by healthy intestinal microflora, which is directly related to the better immune status of the animals [37].
Probiotics are "living microorganisms that, when given in the right amount, bring health benefits to the host" [38]. Supplemented in the right amount, they exert a positive effect on the microbial ecosystem of the host intestine, primarily ensuring a balance between commensal and pathogenic microbiota [39]. The use of probiotic supplements improves bowel function and protects the digestive tract from pathogenic organisms [40]. Some probiotics have shown protective properties against Salmonella [41]. Brisbin et al. (2010) investigated the effect of bacteria normally living in the digestive tract of chickens on the expression of cytokine genes in lymphoid cells [42]. These bacteria were Lactobacillus acidophilus, Lactobacillus reuteri, and Lactobacillus salivarius. Mononuclear cells isolated from cecal tonsils and the spleens of chickens were used in this experiment. The results indicated that mRNA expression of IL-1 was higher in spleen cells than in caecal tonsil cells. In the caecal tonsil cells, there were similarities and differences between the probiotic strains in inducing the mRNA levels of the tested cytokines IL-12p40, IL-10, IL-18, TGF-β4, and IFNγ. L. acidophilus induced higher levels of IFN-γ, IL-12, and IL-1 expression than L. reuteri and L. salivarius, indicating that it has a greater ability to induce a putative Th1 response. L. salivarius inhibited the expression of proinflammatory cytokines and had the unique ability to generate TGF-β, suggesting that these bacteria may trigger an immune response. This experiment provides evidence that Lactobacillus spp. are capable of stimulating spleen and cecal tonsil cells in vitro [42].
Synbiotics are defined as synergistic combinations of probiotics and prebiotics that enhance the positive effects of these products administered alone. One of the functions of synbiotics is to improve the viability of microorganisms in the digestive tract [34]. Sunu et al. (2021) examined a synbiotic of garlic combined with Lactobacillus acidophilus for broilers [43]. After administration of the synbiotic, the intestinal morphology was assessed based on the height of the villi in the duodenum, jejunum, and ileum. The effect of the synbiotic on the immune organs was also assessed and an increased weight of the bursa of Fabricius, thymus, spleen, and caecal tonsil was found. Effects on pH, the number of E. coli, and the number of coliforms were also noted. Giving synbiotics T2 (T2 = basal feed + 4 mL synbiotic) reduced the coliform bacteria. The number of lactic acid bacteria was increased, which is beneficial for its host. The pH of the duodenum, jejunum, and ileum were significantly reduced by the synbiotics. The results showed that this treatment improved many aspects in broiler chickens such as performance, nutrient digestibility, blood profile, and intestinal health [43].

Immunomodulatory Effects of Bioactive Compounds
Plant extracts as well as prebiotics, probiotics, and synbiotics may have immunomodulatory properties. Table 1 summarises the bioactive products and compounds tested in chicken in vitro and in vivo models. Al-Kassie (2009) used thyme and cinnamon to test their effects on broiler performance and biochemical results [44]. This experiment showed that herbal extracts of vegetable oil can affect both performance and the immune system in broiler chickens. The H/L ratio was lowered, and the number of white blood cells increased using only small amounts of this supplement [44]. Other studies have used aqueous and ethanol extracts of Ficus religiosa to test the anti-coccidiosis effects in broiler chickens [45]. Brisbin et al. (2015) investigated the effects of three different Lactobacillus spp. on chicken macrophages in vitro and determined that lactobacilli stimulated the immune response of the macrophage-like cell line MQ-NCSU [46]. The immunostimulating potency of acemannan from Aloe vera was determined in spleen-derived macrophages collected from vaccinated chickens [47]. The effect of probiotic bacteria on immune cells has also been studied using in vivo models. An example of this type of research may be the dietary supplementation of lactobacilli in broiler chickens, which increased cytokine levels and T cell counts [46,47]. The probiotic also triggered an increase in antibody production and intestinal immune responses [48]. Jung et al. (2010) studied a combination of probiotics and fermented herbs and confirmed that the mixture increased the activity of the immune system and helped overcome Salmonella gallinarum infection in chicken [41]. Extracts from herbs and spices contain molecules (flavonoids and terpenoids) that inhibit the metabolism of inflammatory prostaglandins [41]. Supplementation of Aloe secundiflora in chickens increased antibody titres and concentrations of IL6 [49]. 22 commercial broiler chicks of mixed-sex at the age of 5 or 6 weeks. The mononuclear cells of the spleen and the tonsils of the spleen were isolated and cultured The three lactobacilli induced a much higher expression of interleukin 1β in spleen cells than in cecal tonsil cells-more inflammatory response in the spleen than in the cecal tonsil cells. L. acidophilus was more effective in inducing T-helper-1 cytokines, while L. salivarius induced a more anti-inflammatory response.

Immune-Related Primary Cells Cultures
Primary cell cultures are derived directly from freshly harvested tissues and are grown under specific in vitro conditions. They are excellent research models due to their tissue origin, lack of modification, and similarity to the in vivo state. Chicken primary cell cultures are used to study the physiology and biochemistry of cells and the effects of drugs and toxic compounds [54]. One of the disadvantages of primary cells is that they have a limited lifespan. The most common types of primary cells are fibroblasts, keratinocytes, epithelial cells, and mesenchymal stem cells [55].
There are several protocols for the cultivation of primary cells of avian origin that can be used to study the immunomodulatory role of bioactive compounds ( Table 2). One of these primary cell cultures is peripheral blood mononuclear cells (PBMCs). The advantage of PBMCs is that they are easy to access because they come from whole blood. In the study by Husáková et al. (2015), PBMCs isolated from chicken blood were used to study probiotics in vitro [52]. PMBC were used in one experiment to analyze the stimulation of toll-like receptor (TLR) ligands and live probiotics. This experiment enabled the analysis of TLR-mediated cytokine gene expression related to the immune response against substances listed in the table below ( Table 2). The immune response was varied depending on the stimulating substance and the stimulation time [56]. PMBC obtained from ducks was used as cell models for the Duck Plague virus [57].
Intestinal epithelial cells (IEC) have one of the fastest turnovers and renewal abilities among primary cells. IEC enable the analysis of intestinal physiology [58]. IEC have also been used to evaluate the effects of selected Lactobacillus probiotics on the production of avian beta-defensin 9 (AvBD9), which is responsible for maintaining the homeostasis of the gastrointestinal microbiota and the gut immune system [59]. Research on intestinal epithelial cells is increasingly popular due to their association with immunity [60].
Chicken bone marrow-derived dendritic cells (BMDCs) originate from bone marrow cells collected from bones (usually femur and tibia) [61]. They are often used to study the responses of these cells to various types of Salmonella infections or the administration of vaccines. Biggelaar (2021) performed proteomic and RT-qPCR analyses of unstimulated cells using BMDCs and following the administration of the IBV+NDV vaccine, IBV antigen, and lipopolysaccharides produced by E. coli [62]. The RT-qPCR method was used to assess the mRNA expression level of the most elevated proteins in chicken immune cells. This research allowed for the identification of target proteins that might be used to evaluate the quality of inactivated in vitro poultry vaccines [62]. Research carried out by Matulova et al. (2012Matulova et al. ( , 2013 concerned gene expression during Salmonella enterica in-fection [63,64]. These studies have shown the increased traction of genes responsible for inflammation or cytoskeletal regulation and cell migration in the bursa of Fabricius [63,64].   [56] Other primary cell cultures can be determined using mononuclear cells isolated from the spleen or cecal tonsils, which are mainly made up of lymphocytes. Koenen et al. (2004) used splenic mononuclear cells to test for probiotic bacteria. He created an in vitro assay that may be used to screen lactic acid bacteria for immunomodulatory characteristics in chickens in vivo. Strains that have a good impact on spleen lymphocyte proliferation in vitro also have a positive impact on particular humoral immune responses in vivo [65]. Brisbin et al. (2010) established primary cultures of mononuclear cecal tonsil cells in coculture with probiotic bacteria to investigate inflammatory responses [42]. The results of this research are showed below in Table 2.

Immune-Related Cell Lines in Chicken
The cell line is established based on cells from a primary culture that multiply in vitro. Avian cell lines must meet several conditions to be useful for in vitro studies. The first condition is rapid proliferation, which ensures the high efficiency of cells for screening. The cell line should be relatively easy to maintain in vitro. A key condition is that the cell line is homogeneous and free from all kinds of impurities, such as other cell lines or mycoplasma [55]. The growing use of cell lines in poultry and other livestock species is due to their profitability compared to the maintenance of live animals. Cell lines provide an unlimited supply of research material, which makes the results easily reproducible [66]. Table 3 provides an overview of the avian cell lines associated with the immune system. DT40 is a B cell line established from bursa lymphoma caused by leukemia virus infection [67]. DT40 can be used for the screening and selection of synbiotics for chickens [68]. DT40 has also been used to study the molecular basis of immune response in chickens [69]. Dunislawska et al. (2017) analyzed the transcriptional activity of the immune responses against KHL (Keyhole limpet hemocyanin), LTA (lipoteichoic acid) from Staphylococcus aureus, and LPS (lipopolysaccharide) from Escherichia coli. Gene expression analysis showed that KLH and LTA up-regulated forkhead box J1 (FOXJ1) and integrin beta 4 (ITGβ4) in the DT40 cell line [70].
HD11 is a macrophagous chicken cell line derived from chicken bone marrow cells transformed by the avian leukemia virus [71]. HD11 was used to test FOS-inulin's ability to support chicken macrophages in the elimination of Salmonella enteritidis [72]. HD11 was also used to study the effects of organic extracts of milk thistle, turmeric, reishi mushroom, and shiitake on the innate immunity and viability of cancer cells. RT-qPCR was used to quantify interferon-α, IL1β, 6, 12, 15, 18, and the tumor necrosis factor superfamily 15 (TNFSF15). The culture of HD11 with the abovementioned plant extracts improved cell proliferation along with inhibiting the growth of cancer cells compared to control [28].
MQ-NCSU is a macrophage cell line derived from the spleen, used to test (and confirm) the immunomodulatory role of β 1-4 mannobiose [73]. Brisbin et al. (2015) tested the effects of three types of Lactobacillus bacteria on the immune system using MQ-NCSU [46]. LMH is not strictly an immune cell line because it contains poultry liver cells immortalized from hepatocellular carcinoma [74]. However, it can be used to study both the metabolic and immunomodulatory properties of bioactive compounds. For example, Spivey et al. (2014) tested the adhesion of Lactobacillus to epithelial cells using the LMH cell line. Such an experimental approach model predicted gastrointestinal colonization using an in vitro LMH model [54]. Intestinal epithelial cell lines are used to study responses to various bioactive compounds, such as probiotics [55,60]. The new cell line is used in various experiments, such as testing the effects of Salmonella enterica. This cell line is derived from chicken intestinal epithelium cells, and it is a clone of MM-CHiC 8E11 [75].

Ex Vivo Organ Cultures
Ex vivo organ cultures combine in vitro and in vivo studies, as this model is based on fragments of organs or tissues grown in vitro. As such, it mimics the natural reactions of organisms, but under controlled conditions. Ex vivo organ cultures may be used where the type of experiment is not capable of being performed on a living organism. Organoid cultures also allow the cultivation of a suitable in vitro model for different studies. Table 4 describes the ex vivo organ cultures carried out in chickens. Most ex vivo organ cultures of chickens come from the ileum [50,55,60]. The digestive tract is a complex environment that maintains close contact with the host and substances from feed, microorganisms, parasites, and toxins [76]. The intestine is a barrier to external microorganisms and pathogens. It regulates the composition of the microflora inhabiting the intestines. The intestine is lined with a single layer of epithelial, endocrine, and immune cells [77]. Ghiselli et al. (2021) isolated and successfully cultured chicken intestinal epithelium cells, starting with intestinal cell aggregates [60].
Three-dimensional chicken enteroids derived from intestinal embryonic villi and adult crypts were obtained by Nash et al. [78]. These enteroids allow modeling infections with avian pathogens, such as Salmonella typhimurium and influenza A virus. In addition to enteroids made of epithelial cells, it is also possible to obtain intestinal organoids, which consist of both epithelial and mesenchymal cells [79][80][81]. One of the disadvantages of ex vivo organ cultures is that they are not always in agreement with in vivo data. Ex vivo cultures, although they mimic functioning organs, still lack certain gut characteristics [82], including physiological interactions with other parts of the body [83]. The study demonstrated the anti-inflammatory action of hops β-acids and, as a result, the potential immunomodulatory activity exerted on host tissue, since they were able to reduce the expression of proinflammatory cytokines even when an inflammatory producing substance was present (i.e., LPS).

In Ovo Injections
In ovo injection can be another avian research model used to study the immunomodulatory effects of a wide range of bioactive compounds, but also many other substances such as vitamins, minerals, or amino acids. The advantages of in ovo injections include less stress for the injected embryos and less methodological effort, the promotion of early bowel maturation, and improved immune status [88]. It has been proven that in ovo injection of bioactive products and compounds also improves health, chicken development hatchability, and the body weight of the hatched chicks [89]. This method allows broiler chickens to stimulate the early immune response [90]. In recent years, the most popular compounds used in ovo are pro-, pre-, and synbiotics, as well as polysaccharides, such as β-glucan or antimicrobial peptides, which have antibacterial properties against various pathogens [91].
Various studies have shown that there are several possible injection sites: air cell, amniotic sac, directly into the chicken embryo, or yolk sac [92][93][94][95]. The preferred site for in ovo injection in the early stages of embryo development is the air cell, but for injections of vaccines and nutrients, the best time is around day 18 of egg incubation [88]. In every research study, in ovo injection site and time are crucial in maximizing eggs hatchability and chicks quality [96]. In commercial conditions, in ovo injection on day 18 of egg incubation is used in broiler chickens for vaccination, e.g., against Marek's disease or in ovo feeding. Another convenient term dedicated to in ovo stimulation is the day 12 of egg incubation. The injection on the day 12 is influenced by a number of factors resulting from the structure of the embryo and fetal membranes at this stage of development. The chorioamniotic membrane is fully developed and highly vascularized at this stage of embryonic development. As a result, substances that penetrate the membrane enter the blood vessel system and are further transported to the embryo. An additional argument is also the size and arrangement of the embryo in the egg, allowing the noninvasive introduction of the needle into the air chamber without risking puncture. The immunomodulatory properties of the in ovo administration of bioactive substances on day 12 of egg incubation have been extensively described by Siwek et al. (2018) [97].

In Vivo Models vs. In Vitro Models
There are many models of experimental animal studies, including in vivo (live animals) and in vitro (cell and tissue cultures) approaches [98]. In vivo studies are considered the most informative due to the level of complexity of the biological system. Conducting research on live animals allows one to assess the impact of a given experimental factor on production conditions, as well as analyze physiological processes occurring in the environment of the organism, and not in the cell population. On the other hand, they provide confounding factors that make it difficult to interpret the data, for example, in hormonal disorders caused by stress and discomfort [99]. Finally, experiments on live animals involve ethical issues. Laboratories are required to implement 3R strategies (reduction, refinement, and replacement of laboratory use of animals). One of the key elements of the 3R concept is the replacement of experimental animals with in vitro and ex vivo models [100].
Each model has its advantages and disadvantages. In vitro cultures are extremely useful and ethically acceptable, but they are only a simplified model. The ultimate objective of cell or tissue culture is to reflect in vivo conditions as closely as possible. In vitro models are usually used at initial research stages and are followed by experiments on living organisms (e.g., laboratory animals or clinical studies). The results obtained in vitro need to be validated in the living organism, at the presence of various confounding factors. Cell/tissue culture undergo various manipulations, which are not present in natural conditions, such as freezing or thawing cells. On the other hand, cell or tissue cultures allow for more repetitions in a shorter time, reduce interfering environmental factors, and are simpler. For example, one of the applications routinely performed on in vitro cultures is to evaluate the toxicity and efficacy of various compounds on cells [101]. The most basic response of cells is their viability/unprofitability in response to different concentrations of the test medium [102]. The widespread use of cell and tissue cultures is hampered by technical reasons and their instability [98]. Working with primary cells is much more complicated than working with cell lines. Primary cells need more time to grow than cell lines and have limited growth potential. Another aspect is that it is time-consuming to establish the optimal growth conditions for such cells. Cell or tissue cultures are quite costly in respect to the reagents and consumables. There is also need for the laboratory used for cell cultures to maintain sterile conditions, and to be equipped with incubators, microscopes, and laminar chambers.

Future Directions
Nowadays, many factors contribute to the growing interest in bioactive products in poultry farming. This is related to the growing awareness of animal welfare and the food quality and safety. A major reason for turning to natural compounds is the antimicrobial resistance developed by many microorganisms, including human pathogens. Therefore, the line of research revolving around bioactive products with immunomodulatory properties is very promising. The cell culture models that have been recently developed are definitely more complex, so that they can closely mirror the in vivo model. Such an approach facilitates the studying of the effects of the bioactive compounds and products, and the results can be more directly applied to improve the health status of animals. The directions for further development of this line of research is to search for natural alternatives to antibiotics and therapeutic compounds, e.g., against avian influenza [103].

Conclusions
Different types of bioactive products, such as herbs, spices, plant extracts, as well as prebiotics, probiotics, and synbiotics have been used as supplements in poultry due to their immunomodulatory effects. The development of new supplements that improve performance and health in poultry can be facilitated by the use of various biological models. There are several in vitro and ex vivo models that can be used to study the immunomodulatory role of bioactive compounds in poultry. Additionally, an in ovo model allows for the injecting of the tested products directly into the structures of the developing embryo. This article provides a detailed overview of the available protocols or models that can be applied in immune-related studies to assess the specific properties of bioactive compounds in poultry prior to animal studies.