The global poultry industry holds immense significance in meeting the continuously increasing demand for high-quality protein-rich food, driven by the continuous rise in the global population [1]. To achieve optimal growth and productivity of the birds, ensuring their health and well-being is essential. 

The intestines are responsible for the digestion and absorption of essential nutrients from the feed consumed by poultry [2]. Furthermore, the intestinal tract is immensely important in the immune system of the animals. It serves as the physical barrier and also hosts abundant organized lymphoid tissue and immune effector cells, which collectively provide protection against pathogens and toxins ^[3][4][5][3,4,5]. Nevertheless, the intestines possess the most extensive exposed surface in the body [6]. The constant exposure to a wide range of potentially harmful substances makes them susceptible to many diseases, such as coccidiosis and necrotic enteritis, which have a significant impact on the poultry industry ^[7][8][7,8]. Maintaining a healthy intestinal tract has been a focus of research for decades, and this area is now gaining more attention due to the growing awareness of animal welfare, food safety, and the increasing public scrutiny towards the use of antibiotic growth promoters [9].

2. Methionine and Arginine in Intestinal Health

2.1. Methionine and Arginine in Intestinal Development and Repair

The intestinal epithelium, which comprises a single layer of epithelial cells and a protein-rich mucus layer, has one of the highest turnover rates in the body ^[10][56]. The renewal of epithelial cells in the intestinal epithelium involves a highly coordinated process of cellular proliferation, differentiation, migration, and apoptosis. This constant cell turnover relies on ongoing protein synthesis, which is largely mediated through the activation of mTOR pathway ^[11][57]. In addition to serving as essential building blocks of proteins, Met and Arg have been shown to activate mTORC1 ^[12][13][58,59]. Met activates the mTOR signaling pathway by promoting the methylation of phosphatase 2A through the action of SAM. Furthermore, previous studies showed that SAM could bind to the S-adenosylmethionine sensor upstream of mTORC1 (SAMTOR), counteracting its inhibitory effects on mTORC1 activity ^[13][14][59,60]. On the other hand, Arg activates the mTOR pathway through inhibiting the activity of tuberous sclerosis complex 2, a protein that normally suppresses the mTOR pathway ^[15][61]. The mTOR pathway is essential for maintaining intestinal epithelial cell proliferation during both homeostasis and regeneration, as research showed that a disruption in this signaling pathway would lead to intestinal epithelial cell defects and hinder the intestinal regeneration ^[16][17][62,63]. The Wnt/β-catenin signaling pathway is also well established for its role in maintaining intestinal structure and homeostasis as it regulates the self-renewal and differentiation of the intestinal stem cells ^[18][19][20][64,65,66]. Interestingly, studies have also shown that both Met and Arg can activate the Wnt/β-catenin pathway to enhance the intestinal epithelial development ^[21][22][32,67]. Met is required for the sequestration of glycogen synthase kinase 3, which is an essential step in activating the Wnt signaling pathway ^[23][68]. The modulating effects of Arg on the Wnt/β-catenin signaling pathway can be attributed to the synthesis of NO, which is a known activator of this pathway ^[24][69]. Intriguingly, activation of the Wnt/β-catenin signaling pathway is closely regulated by the methylation of the Arg residues in Ras GTPase-activating protein-binding protein 1 (G3BP1) with SAM as the methyl donor. This linkage underscores the collaborative regulatory roles of Met and Arg in this pathway ^[23][25][68,70]. The activation roles of Met and Arg in both pathways contribute to the regenerative capacity and development of the intestine. Beyond their roles in pathway activation, Met and Arg are indispensable for polyamine synthesis. Arg serves as the precursor for polyamine synthesis, while SAM, a product of Met metabolism, functions as the methyl donor in this process ^[26][71]. Polyamines are essential for intestinal epithelial renewal and repair, ensuring its important roles in cell proliferation, development, and migration ^[27][28][72,73]. Studies have demonstrated that providing dividing cells in the crypts with polyamines can stimulate mucosal growth and facilitate the repair of damaged mucosa ^[29][30][31][74,75,76]. The proposed mechanism underlying the stimulation effect of polyamines in mucosal growth involves their ability to regulate expression of various genes encoding growth promoting and inhibiting factors ^[26][32][71,77]. Polyamines are also shown to be vital for the expression of tight junction and adhesion junction proteins which maintain the intestinal barrier function ^[33][34][78,79]. Overall, Met and Arg are essential for the development and repair of the intestinal epithelium. They contribute to this process by activating pathways essential for intestinal regeneration and by participating in polyamine synthesis.

2.2. Antioxidant Effects of Methionine and Arginine on Intestinal Health

2.2.1. Oxidative Stress

The integrity of the intestinal barrier can be disrupted by various factors, among which oxidative stress is a significant contributor. Reactive oxygen species (ROS) are byproducts generated during normal metabolic processes ^[35][36][80,81]. Under normal conditions, the production of ROS is balanced by the antioxidant system ^[37][82]. However, an excessive production of ROS or a decline in antioxidant defenses can disrupt this equilibrium, leading to the accumulation of ROS. The highly reactive ROS will react with the cellular components causing cellular damage, dysfunction, and apoptosis, which ultimately leads to impaired organ functions and the development of oxidative stress ^[35][80]. Several factors during poultry production can cause oxidative stress to the birds, such as nutritional factors like nutrient imbalances and feed toxins, environmental factors like heat stress and stocking density stress, as well as pathological factors ^[38][39][40][83,84,85]. Oxidative stress can damage the structure and function of tight junctions, resulting in compromised intestinal barrier functions and increased permeability ^[41][86]. Oxidative stress is also known to damage the intestinal epithelial cells directly, leading to further disruption of the barrier function and inflammation. The recruited macrophage and heterophils intensify the production of ROS, stimulating a positive feedback loop that exacerbates oxidative stress and inflammation ^[42][43][87,88]. Oxidative stress is also reported to cause morphometric changes in the intestinal tract by reducing the villi height and lowering the villus: crypt ratio, interfering with nutrient absorption ^[44][45][89,90].

2.2.2. Antioxidant Effects of Methionine and Arginine

Methionine is reported to exhibit potent antioxidant capacity through two major mechanisms ^[46][47][91,92]. Firstly, it produces antioxidant metabolites by undergoing metabolic processes. SAM, as one of the metabolites in the pathway, is not only an important methyl donor, but also a key metabolite modifying antioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT) ^[46][47][91,92]. Such modification effects were confirmed by different research groups observing increased activities of antioxidant enzymes in birds fed Met supplemented diets ^{[48][49][50][51][52]}[93,94,95,96,97]. Cysteine, another sulfur-containing amino acid, also exhibits antioxidant ability and is further metabolized to GSH, a well-known intrinsic antioxidant. Previous studies have also demonstrated increased GSH or improved GSH: glutathione disulfide (GSSG) ratio in broilers fed Met supplemented diets ^[39][53][54][84,98,99]. Secondly, Met residues in proteins can directly scavenge ROS. As a sulfur-containing amino acid, Met residues on the surface of proteins are readily oxidized. By scavenging ROS and being oxidized into methionine sulfoxide, Met can protect other critical components from oxidation, thus maintaining their integrity and function ^[25][55][17,70]. Furthermore, methionine sulfoxide can be reduced by methionine sulfoxide reductases (MSR) back to Met to restore its antioxidant capacity ^[56][57][58][20,100,101]. The antioxidant capacity of methionine and its metabolites is presented in Figure 1. Previous research has provided evidence on the beneficial effects of Met supplementation in intestinal health through its antioxidant capacity. Dietary Met supplementation in broilers improved the GSH:GSSG ratio and glutathione peroxidase activity as well as increased the villus height (VH) and ratio of villus height to crypt depth (VH:CD) with or without stocking density challenge ^[59][29]. Met supplementation also improved the tight junction protein expression, whereas it decreased the expression of proinflammatory cytokines in broilers under heat stress ^[60][30]. Another study also demonstrated that Met supplementation increased the concentration of GSH, while it reduced the malondialdehyde (MDA) content in the duodenum mucosa. The VH and VH:CD were again improved by Met supplementation ^[61][31]. Despite not being widely recognized for its antioxidant capacity, several studies have shown that Arg supplementation can improve the oxidative status and benefit intestinal health. In an in vitro study conducted in ovine intestinal epithelial cells ^[62][38], the researchers found that Arg supplementation significantly reduced the hydrogen peroxide-induced ROS production. Furthermore, Arg also increased levels of glutathione peroxidase and tight junction protein 1, whereas it decreased the level of proinflammatory cytokines. Moreover, supplementing Arg in broiler breeders improved the oxidative status of the breeder birds as well as the one-day-old offspring ^[63][37]. Another study conducted in rats challenged with sodium nitrite showed that Arg supplementation reduced the serum MDA content and increased GSH production ^[64][102]. Additionally, both Met and Arg have been shown to activate the nuclear factor erythroid 2-related factor 2–antioxidant responsive element (Nrf2-ARE) pathway, leading to the upregulation of antioxidant enzymes and the generation of antioxidant effects ^[65][66][103,104]. Considering the potent antioxidant capacity and effects of Met and Arg, incorporating both Met and Arg as functional dietary supplements for poultry holds significant promise in mitigating the detrimental effects of oxidative stress on intestinal health and the overall well-being of the birds.

2.3. Methionine and Arginine in the Immune System

The intestinal tract is a major compartment of the immune system. The gut-associated lymphoid tissues (GALT) are estimated to comprise more immune cells than any other tissues ^[67][105]. The definable structures of GALT include lymphoid aggregates located within the lamina propria, Meckel’s diverticulum, Peyer’s patches, cecal tonsils, and bursa of Fabricius. In contrast to mammals, birds lack traditional lymph nodes; however, they possess numerous lymphoid aggregates that represent most of the secondary lymphoid tissues. Overall, the gut harbors diverse immune cell types, including heterophils, macrophages, dendritic cells, natural killer cells, and B and T cells, with the cellular compositions differing among different lymphoid tissues ^[68][106]. Methionine plays a critical role in both the humoral and cell-mediated immune responses in animals ^[69][107]. The impact of methionine on the humoral immune function of animals is primarily reflected in its effects on immunoglobulin levels in the body ^[70][108]. As for the cell-mediated immune responses, Met exerts regulatory effects on T cell activation and development ^[71][109]. Research showed that the activation of T cells is typically associated with an upregulation of Met transporters and SAM metabolism enzymes ^[72][110]. It was proposed that Met as well as its metabolites are taken up by the T helper cells for the synthesis of new protein and methylation of RNA and DNA, which drives activation, proliferation and differentiation of T cells ^[71][73][109,111]. Sufficient levels of methionine can significantly enhance antibody production as well as improve T cell proliferation in broilers ^{[70][74][75][76]}[108,112,113,114]. Conversely, a deficiency of methionine significantly reduces the levels of immunoglobins in the bloodstream and inhibits the proliferation and differentiation of lymphocytes in broilers ^[77][78][115,116]. Numerous studies have extensively explored the immunoregulatory roles of Arg. Arg plays a significant modulatory role in immune function, primarily through the synthesis of NO by inducible NOS (iNOS) in macrophages ^[79][80][117,118]. NO is a versatile molecule that serves as a pivotal mediator in various immunological processes, including antimicrobial defense, immune cell regulation, and cytotoxicity ^[81][82][119,120]. Research has also shown its protective effects against protozoan infections ^[83][84][121,122]. Over the years, researchers have also shown that arginine availability is crucial for maintaining normal T cell proliferation and function. Rodriguez et al. ^[85][123] demonstrated that T cell function was significantly impaired under conditions of limited arginine supply, while this effect was completely reversed when arginine was replenished. It was proposed that arginine is indispensable for the expression of the T cell antigen receptor CD3ζ, which subsequently influences T cell function ^[85][86][25,123]. A recent study has also reported that Arg supplementation ameliorated the negative effect in Eimeria-infected broiler birds by enhancing the T cell function and elevating NO production ^[87][21]. Given the important roles of Met and Arg in immunity, the supplementation of Met and Arg could be a promising approach to enhance the immune response of the poultry intestine that constantly faces various challenges.

2.4. Methionine and Arginine in the Intestinal Microbiome

Numerous bacterial species inhabit the gastrointestinal tract (GIT) of chickens, with Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria being the dominant ones at the phylum level ^[88][124]. Studies have revealed that maintaining a well-balanced microbiota profile is crucial for preserving intestinal health and normal functions ^[89][125]. For instance, these microbial communities break down polysaccharides to provide amino acids and short-chain fatty acids, serving as crucial energy sources for the epithelial cells ^[90][126]. Moreover, the healthy intestinal microorganism plays a protective role against pathogens by inhibiting the adherence and colonization of the pathogens as well as modulating the immune responses of the host ^[88][91][124,127]. The intestinal microbiota profile is known to be influenced by dietary factors ^[92][128]. Recent research has shed light on the impact of Arg supplementation on the intestinal microbiota of chickens. Multiple studies have consistently shown that Arg supplementation can lead to an increase in the relative abundance of Firmicutes and a decrease in Proteobacteria in the ileum ^[93][94][95][34,35,129]. At the genus level, Ruan et al. ^[94][35] demonstrated that Arg supplementation resulted in a higher relative abundance of Romboutsia and a lower abundance of Clostridium sensu stricto 1. Brugaletta et al. ^[96][130] found that in the ceca, Arg supplementation decreased alpha diversity and the relative abundance of Proteobacteria, while increasing the relative abundances of Bacteroidetes and Lactobacillus salivarius. There is limited evidence regarding the effects of Met supplementation on the chicken intestinal microbiota profiles. However, Kumar et al. ^[97][131] reported that Met supplementation could enhance glycolysis and energy generation by the cecal microbiota. Another study performed in germ-free pigs revealed that in the absence of intestinal microbiota, Met levels in the ileum and hindgut increased significantly, indicating that the intestinal microbiota were actively involved in Met metabolism ^[98][132]. In conclusion, the findings so far suggest that Arg supplementation has notable effects on the intestinal microbiota composition in chickens. However, further investigation is needed to explore the potential impacts of Met supplementation on the chicken intestinal microbiota.