Roles of functional amino acids in the immune system of pigs

Key information

Amino acids are involved in various important metabolic pathways beyond growth, including modulating the functioning of the body’s immune system.
During a condition of sub-clinical disease, increased dietary supply of functional amino acids (i.e. methionine, tryptophan, threonine, arginine, glutamine, and glycine) ameliorates the negative effect of growth reduction associated with immune challenge.
Based on the available literature, increasing ideal ratios of some functional amino acids – such as the standardized ileal digestibles methionine+cystine:lysine (+ 6%-points), tryptophan:lysine (+ 3%-points), and threonine:lysine (+ 5%-points) – relative to the ratios applied for their healthy counterparts can enhance immune status and optimize growth performance of pigs challenged by sub-clinical disease.
In pigs with subclinical infections of the gut, increased dietary supply of threonine, arginine, glutamine, and glycine may help enhance immune status and gut integrity.
Further research is needed to assess the interaction or synergistic effects of these functional amino acids on an animal’s immune function.

Introduction

Pigs are often exposed to chronic subclinical levels of diseases and environmental stress in commercial farms, resulting in a reduced feed intake and impaired performance. Differences in the health or immune status of pigs are one reason for the large variation in their performance among commercial farms (Pastorelli et al., 2012). The feeds provided to the animals must supply the optimal levels of nutrients, including amino acids (AAs), if growth, health, and productivity are to be maintained at optimal levels. The immune system is a defense system to protect the host from invading pathogens that can take advantage of metabolic or digestive disturbances, resulting in pathogen proliferation and immune system activation.

The gastrointestinal tract (GIT; from the stomach to colon) is a key component of the body's systemic immune system. In addition to digestion and absorption of nutrients, it serves as an immunological barrier by secreting digestive enzymes and hormones from the enterocytes. When speaking about gut immunity, the focus is mainly on weaned piglets because of their immature digestive and immune capacity associated with a greater incidence of gut disorders such as post-weaning diarrhea. Both systemic and gut immune challenges increased after the 2006 ban of antimicrobial growth promoters (AGPs) in animal feeds in the European Union (EU).

Various feed additives are used to enhance the immune status of the animals, including probiotics, prebiotics, organic acids, essential oils, phytogenics, enzymes, antibiotics (in some countries), and AAs. Some functional AAs are involved in immune system functioning by regulating 1) the activation of T lymphocytes, B lymphocytes, natural killer cells, and macrophages, (2) the cellular redox state and gene expression, and (3) the production of antibodies, cytokines, and other cytotoxic substances (Li et al., 2007). During immune system stimulation (ISS), nutrients are redirected away from growth and towards tissues involved in immune response (Reeds and Jahoor, 2001). This implies that the production of compounds involved in the immune response will require more of some key AAs. Thus, increasing dietary levels of some functional AAs is a possible solution for maintaining gut health and promoting growth, as these AAs can enhance immune status and gut integrity in weaned pigs. This is particularly important when sanitary and environmental conditions are challenging and the AGPs are not included in the diet.

The intent of this article is to review the immune system of pigs, the role played by functional AAs in that immune system, and their application to improving immunological capacity during immune challenge conditions. For a more in-depth review of the effects of AAs on the immunity of animals, see Li et. al. (2007) and Wu (2010).

Immune system of pigs

The immune system is an adaptive defense system to protect the host from invading pathogenic microorganisms, i.e. bacteria, viruses, fungi, and parasites (Kuby, 1994). There are two components of the immune system: innate (nonspecific) and acquired (specific) immunity (Figure 1). Innate immunity is the basic initial protection against infection and comprises four types of defensive barriers: 1) anatomic (skin), 2) physiologic (temperature, pH, oxygen tension), 3) phagocytic (ingestion of macromolecules by macrophages), and 4) inflammatory (Kuby, 1994). The innate immune system consists of the cellular components, which include monocytes, macrophages, dendritic cells, neutrophils, natural killer cells, and extracellular mediators such as cytokines, chemokines, acute phase proteins, the complement system, epithelial barriers, and antimicrobial peptides (Parkin and Cohen, 2001; Tan et al., 2013).

Acquired or specific immunity is induced by exposure to an antigen, naturally or via vaccination, and developed more slowly (Kuby, 1994). The acquired immune system can be further divided into humoral and cell mediated immunity. During the humoral immune response, B lymphocytes in the blood secrete immunoglobulins (antibodies) to bind and eliminate foreign antigens (Parkin and Cohen, 2001). The cell-mediated immunity functions as the interaction of T cells (T lymphocytes) and their associated cytokines to eliminate intracellular antigens. Two major types of T cells are T helper (CD4+ Th) and cytotoxic T cells (CD8+). The CD4+ Th cells recognize foreign antigens and assist other cells to eradicate the pathogen. The CD8+ cytotoxic cells are involved in antiviral and antitumor activity (Parkin and Cohen, 2001).

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Figure 1 Overview of immune system in pigs (Adapted from Kampman – van de Hoek, 2015)

Gut immunity

The GIT, i.e. from the stomach to colon, is an important component of the body's immune system because it contains more than 10¹² lymphocytes and has a greater concentration of antibodies than any other site in the body (Mayer, 2000). In addition to the digestion, absorption, and metabolism of nutrients, the intestinal epithelial cells (enterocytes) secrete digestive enzymes and hormones and form an interface between the external environment (e.g. dietary nutrients, microbes, pathogens, and toxic compounds) and the animal, serving as an immunological barrier (Stoll and Burrin, 2006). The GIT also serves as a home for various microbes, which synthesize and utilize nutrients. Thus, the GIT is one of the body’s most metabolically active and complex tissues.

The intestinal epithelial cells also participate in the innate immune system of the GIT by their ability to secrete mucus and antimicrobial peptides (Shao et al., 2001). The mucus layer is mainly composed of mucins, which are glycosylated proteins secreted along the epithelium of the GIT to protect the gut wall from damage and maintain immune function (Li et al., 2007). Antimicrobial peptides (defensins and cathelicidins) and immunoglobulins secreted by epithelial cells act in concert to restrict the interaction of potential pathogens with the gut mucosa (Oswald, 2006). Furthermore, the epithelial cells can produce cytokines such as interleukin (IL)-1, IL-10, IL-15, and IL-18, as well as chemokines, which are crucial for the recruitment and activation of immune cells (Stadnyk, 2002).

When referring to gut immunity, the focus is mainly on weaned pigs because their digestive function is not fully developed and is more susceptible to pathogens and immune challenges than that of mature pigs. In newborn pigs, antibodies in the sow’s colostrum and milk provide the first source of immune protection. The neonatal pig is immunologically immature until about 4 weeks of age (Blecha, 2001). Currently, piglets are weaned at 3 to 4 weeks of age under commercial conditions. The first few days after weaning are a stressful time and often associated with reduced feed intake and growth, impaired intestinal barrier function, and increased incidence of diarrhea due to immature digestive and immune systems (Pluske et al., 1997; Moeser et al., 2007). Therefore, the development and maintenance of gut immunity is crucial not only for the innate immune defense of the newly weaned pigs but also for the development of the overall mature immune system and performance in later growth stages.

Modulation of immune challenge on nutrient utilization

Pigs exposed to a sub-clinical state of disease in commercial farms have a lower nutrient utilization and performance than what is potentially possible under good conditions (Colditz, 2002). A review by Pastorelli et al. (2012) reported that immune challenges – especially bacterial infections of the GIT – reduced the body weight (BW) gain of growing pigs by as much as 40%, which was partly due to the reduction in feed intake. The decreased feed intake and BW gain are associated with systemic inflammation, which is brought about by pro-inflammatory cytokines released by the innate immune system (Li et al., 2007). Pro-inflammatory cytokines, particularly IL-1β, IL-6, and tumor necrosis factor (TNF)-α produced by stimulated macrophages, modify nutrient utilization during an immune challenge and have a direct effect on the liver, brain, muscle, and fat tissue (Colditz, 2002).

During ISS, nutrients are redirected away from growth and towards tissues involved in immune response (Reeds and Jahoor, 2001). Under such conditions, the liver becomes the major contributor to the whole body protein synthesis, largely because of increased production of a wide range of acute phase proteins (APP), while synthesis and muscle protein gain are reduced. Major APPs in pigs include haptoglobin, fibrinogen, C-reactive protein, serum amyloid A, porcine major APP, and albumin (Chen et al., 2003). These changes in the rate and type of protein synthesis will have a direct impact on AA needs at the tissue level, both qualitatively and quantitatively (Reeds and Jahoor, 2001; Obled, 2003).

Immune cells utilize AAs to maintain clonal proliferation, but more importantly the liver also needs AAs for gluconeogenesis as well as the synthesis of APP and glutathione, which are essential for immune function (Hunter and Grimble, 1994; Reeds and Jahoor, 2001). The immune status of animals greatly depends upon the availability of AAs and other substrates for the synthesis of these proteins and peptides (Li et al., 2007). The AA profile required for immune function is different from that of AAs required for muscle protein deposition (Reeds and Jahoor, 2001). During ISS, Lys that is typically first limiting for growth will be in relative excess, whereas other AAs used by immune cells (e.g., Met+Cys, glutamine, tryptophan, and threonine) may become limiting (Reeds and Jahoor, 2001). As a result, AA requirements for immune functions are not the same as for optimal growth performance. More closely meeting AA requirements for maintaining good immunity during ISS (e.g. sub-clinical level of disease) will reduce the negative impact of ISS on animal performance, thereby improving production efficiency.

Roles of functional amino acids in the immune system

In addition to their primary role of serving as a building blocks for protein synthesis, AAs are involved in various important metabolic pathways in the body. Amino acids that regulate key metabolic pathways to improve the health, survival, growth, development, lactation, and reproduction of organisms are defined as functional AAs (Wu, 2009). Indeed, some functional AAs modulate immune system functions in the GIT, thymus, spleen, lymph nodes, and immune cells of the circulating blood (Cunningham-Rundles, 2002). In general, AAs influence the immune system by enhancing immune status to prevent infections and reducing or eliminating established infections such as inflammation and autoimmunity (Yoneda et al., 2009). Since the EU ban on AGPs, the roles played by functional AAs in the immune system have received more attention. The most important functional AAs include sulfur amino acids, i.e. methionine and cysteine (SAA; Met and Cys), tryptophan (Trp), threonine (Thr), glutamine (Gln), arginine (Arg), and glycine (Gly; Li et al., 2007). The following section summarizes the roles and beneficial effects of dietary supplementation with these functional AAs, with a focus on swine diets.

Sulfur-containing amino acids

Methionine is a nutritionally essential AA, while Cys is a nonessential AA because it can be synthesized from Met but cannot be transformed into Met. Methionine serves as a methyl donor for important processes such as DNA methylation and polyamine synthesis (Grimble, 2002), a role that becomes increasingly important during immune challenge to enhance the proliferation of immune cells (Dwyer, 1979). Cysteine is the rate-limiting substrate for the synthesis of glutathione(GSH), which is the major intracellular antioxidant, consisting of a tripeptide of glutamate (Glu), Cys, and Gly (Wu et al., 2004). Glutathione occurs in a reduced (GSH) and disulfide-oxidized (GSSG) form within the cell, and the GSH:GSSG ratio indicates the reduction/oxidation (redox) potential (Roth, 2007). An increased redox potential is an indication of improved immune status, as a decreased GSH:GSSG ratio is associated with cellular oxidative stress (Fang et al., 2002) and intestinal atrophy in pigs (Wang et al., 2008). Besides functioning as a scavenger of free radicals and other reactive oxygen species (ROS), GSH is involved in immune functions, as it is needed for the activation of T-lymphocytes and leukocytes and for the production of cytokines (Lu, 2009; Wu et al., 2004).

Cysteine is also needed to produce taurine, which acts as a cell membrane stabilizer and antioxidant (Grimble, 2002) and is particularly abundant in leucocytes (Roth, 2007). The synthesis rate of fibrinogen, an APP containing about 4% Cys, is increased by approximately 140% in pigs with immune activation as a result of a turpentine injection (Jahoor et al., 1999). During ISS, the utilization of Cys for the production of compounds that are involved in the immune response – such as GSH, taurine, and APP – is increased (Grimble, 2002). This implies that the need for Met and Cys also increases during situations of immune challenge. The following paragraph describes the better use of Met to supply the additional Met+Cys requirement.

Rakhshandeh et al. (2010) reported that ISS by injection of lipopolysaccharide (LPS), components of the cell wall of E. coli strain, did not demonstrably affect the ileal digestibility of AAs and energy. However, LPS stimulation reduced the ratio of whole-body nitrogen (N) and sulfur (S)-balance, indicating that SAAs are preferentially preserved for the production of non-protein compounds such as glutathione to enhance immune status. In another study, Rakhshandeh et al. (2014) found that ISS reduced whole body protein deposition and decreased the daily SAA requirement (Figure 2). However, ISS increased maintenance SAA requirements: For example, to achieve a constant protein deposition of 50 g/d, unchallenged (ISS-) pigs need 1.63 g of standardized ileal digestible (SID) SAA intake, while immune-challenged (ISS+) pigs need 1.87 g SID of SAA intake (i.e. a 15% increase).

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Figure 2 Impact of immune system stimulation (ISS) and standardized ileal digestible (SID)

The concentration and fractional synthesis rate of plasma albumin decreased when LPS immune-challenged pigs were fed a diet low in Met+Cys compared with pigs fed an adequate Met+Cys diet (Litvak et al., 2013a). In a N-balance study with growing pigs, Litvak et al. (2013b) showed that ISS by LPS injection reduced protein deposition rate, while the optimal dietary Met to Met+Cys ratio for maximum body protein deposition increased from 57 to 59% (Figure 3). The increased need of dietary Met+Cys for production of glutathione during ISS is better provided via increased Met supply. Cysteine is extremely unstable and rapidly oxidizes to cystine, resulting in free radical generation that gives Cys its toxic properties (Grimble, 2002). Due to these toxic properties, Cys is predominantly preserved in a dimerized form (cystine) in extracellular fluids, while within the cells, the reduced form of Cys is maintained at relatively low levels sufficient for essential functions such as the incorporation of free Cys into protein and GSH (Stipanuk and Ueki, 2011).

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Figure 3 Protein deposition (g/d) at varying levels of Met:Met+Cys without and with LPS challenge (Litvak et al., 2013b)

The dietary SID Met+Cys:Lys needed to maximize body protein deposition increases from 55 to 75% when growing pigs are immune challenged with LPS (Kim et al., 2012). Under commercial conditions (i.e. prone to pathogens and without in-feed antibiotics), the performance of 25-50 kg pigs was maximized at a SID Met+Cys:Lys ratio of 62.3% based on regression (Zhang et al., 2015; Table 1), which is higher than the current NRC (2012) recommendation of 56%. This agrees with Capozzalo et al. (2014), who reported that the BW gain and feed conversion ratio (FCR) of 8-20 kg pigs infected with E. coli and fed antibiotic-free diets optimized at the SID Met+Cys:Lys ratio of 62.2%. These results indicate that the needs of Met+Cys, including the Met requirement for converting to Cys, are increased during immune challenge.

Table 1 Effect of dietary SID Met+Cys:Lys ratio on the performance of growing pigs (Zhang et al., 2015).

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Tryptophan

In addition to being involved in protein synthesis and serotonin regulation, Trp is important for immune function modulation through the kynurenine pathway, which is initiated by two enzymes. The enzyme tryptophan-2, 3-dioxygenase (TDO) regulates the concentration of homeostatic plasma Trp in the liver. Another enzyme, indoleamine-2, 3-dioxygenase (IDO), which is present in various body tissues (intestine, stomach, lungs, brain) and macrophages, is induced by inflammatory cytokine IFN-γ during immune activation (Widner et al., 2000). More than 95% of dietary Trp, not utilized for protein synthesis, is metabolized through the kynurenine pathway, forming various products such as kynurenic acid and niacin (Botting, 1995).

Studies have shown that lung inflammation in pigs reduces plasma Trp levels (Melchior et al., 2004; Figure 4) and increases IDO activity in lungs and associated lymph nodes (Le Floc’h et al., 2008) compared to pair-fed healthy piglets. Furthermore, they observed that piglets fed a low-Trp diet had a higher plasma concentration of the major APP haptoglobin (which has a relatively high Trp content) than pigs fed a Trp-adequate diet. These results suggest that Trp catabolism via the kynurenine pathway is increased for synthesis of APP during ISS, which may increase the Trp necessary to maintain growth. Furthermore, Trp can be used to synthesize the neurohormone melatonin, which may act as a free radical scavenger and possess antioxidant properties (Le Floc'h and Seve, 2007).

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Figure 4 Plasma tryptophan concentration in pigs with lung inflammation (CFA, complete Freund’s adjuvant) and control (CON) healthy pair-fed weaned pigs (Melchior et al., 2004)

Rearing pigs under poor sanitary conditions can induce a moderate inflammatory response. Le Floc’h et al. (2007) reported that the optimal feed intake and BW gain of weaned pigs kept under poor sanitary conditions were achieved at a higher SID Trp:Lys ratio (21 vs. 18%) than in those kept under good sanitary conditions (Figure 5). The efficiency of Trp utilization for whole body protein deposition of growing pigs was reduced during LPS challenge due to increased usage of Trp for immune functions, and a greater dietary Trp level (7% increase) was needed to maintain body protein deposition at levels similar to those of healthy pigs (de Ridder et al., 2012).

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Figure 5 Effects of sanitary status and Trp:Lys ratio (%) on feed intake and weight gain of growing pigs (Le Floc’h et al., 2007)

Supplementing a diet that contains no in-feed antibiotics with a relatively high level of L-Trp (SID Trp:Lys ratio of 22%) maximized the growth performance of 25-50 kg pigs raised under commercial conditions (Zhang et al., 2012; Table 2). More recently, Jayaraman et al. (2015) also reported that in weaned pigs challenged with E. coli K88 mRNA expression of the pro-inflammatory cytokine TNF-α in ileal tissue linearly decreased with an increasing SID Trp:Lys ratio. Pig performance was optimized at an average SID Trp:Lys of 22.6%. This is in line with Capozzalo et al. (2015), who reported that increasing the dietary SID Trp:Lys ratio to 24% improved FCR and increased the plasma levels of Trp and kynurenine in weaned pigs fed antibiotic-free diets, regardless of infection with E. coli. When weaned pigs housed in a commercial farm were exposed to subclinical diseases and fed antibiotic-free diets, supplementing a higher dietary SID Trp:Lys ratio to 24% optimized BW gain and FCR (Capozzalo et al., 2013).

Table 2 Effect of dietary SID Trp:Lys ratio on the performance of growing pigs (Zhang et al., 2012)

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Threonine

Threonine plays a key role in immune function through its incorporation into immunoglobulins (also known as antibodies), which are produced by plasma cells in response to immune challenge. Threonine is the most prevalent AA in immunoglobulins (i.e. approximately 10% in milk immunoglobulins; Bowland, 1966). More than 60% of dietary Thr is used up in the first pass metabolism of the portal-drained viscera (PDV; includes small and large intestine, stomach, pancreas, and spleen) in pigs (Stoll et al., 1998). Indeed, the Thr requirement of parenterally-fed piglets (i.e. bypassing the first pass by the PDV) was reduced by 55% compared with orally-fed piglets (Bertolo et al., 1998).

The synthesis of mucosal proteins in the GIT includes proteins that are secreted into the lumen, including mucins, which protect the gut from injury and pathogens. Mucins are particularly rich in Thr, which represents about 30% of the total AAs of mucins and 11% of the total endogenous protein in ileal digesta of pigs (Lien et al., 1997). Mucin proteins are continuously synthesized and are resistant to digestion because they contain a high density of O-linked oligosaccharides (Strous and Dekker, 1992). This mean that an increase in mucus secretion will directly increase endogenous losses of AAs, particularly Thr. Thus, Thr is a key AA for the integrity and immunity of the GIT.

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Figure 6 Effect of dietary Thr:Lys ratios on the growth and serum IgG concentration of piglets(Wang et al., 2006)

Cuaron et al. (1984) demonstrated that sows fed a diet adequate in Thr (sorghum and L-Thr based) had 20% more IgG in their plasma than sows fed the Thr-deficient diet at farrowing. Supplementing a low-protein diet with 0.14% L-Thr during gestation increased milk IgG concentration at farrowing and during lactation (Hsu et al., 2001). Furthermore, supplementation with L-Thr to produce a dietary Thr level higher than for optimal growth (i.e. a Thr:Lys ratio of 99%) increased the production of serum IgG and bovine serum albumin antibody levels in 17-31 kg pigs challenged with bovine serum albumin injection (Li et al., 1999). Similarly, Wang et al. (2006) reported that in 10-25 kg pigs challenged with ovalbumin injection, the serum IgG concentration was maximized at a dietary SID Thr:Lys of 78%, while optimal growth performance was achieved at a SID Thr:Lys of 69% (Figure 6).

These results indicate the role of Thr in modulating immune function through its incorporation into immunoglobulin. A farm’s sanitary conditions can affect the health or immune status of the animals. Indeed, Bikker et al., (2007) reported that the SID Thr:Lys ratio to optimize BW gain was higher at 71% for 25-110 kg pigs fed AGP-free diet than in those fed an AGP-added diet, wherein BW gain was maximized at 65% SID Thr:Lys (Figure 7). Jayaraman et al. (2014) also reported that while poor sanitary conditions reduced the growth rate, increasing SID Thr:Lys to 71% could improve gain:feed in piglets fed antibiotic-free diets.

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Figure 7 Effect of dietary SID Thr:Lys ratios on average daily gain (ADG) of 25-110 kg pigs fed diets with or without AGP (Bikker et al., 2007)

Inadequate dietary Thr supply to piglets caused an increased incidence of diarrhea, decreased mucosal weight, and mucin secretion along the GIT (Law et al., 2007), and reduced villus height and villus height to crypt depth ratio in the ileum (Hamard et al., 2007). Wang et al. (2007) found that the fractional synthesis rates (FSR) of jejunal mucosa and mucins were higher in weaned pigs fed the diet with the adequate Thr level (0.74% SID Thr) than in pigs that were pair-fed diets containing an excess (1.11% SID Thr) or deficiency (0.37 % SID Thr) of Thr.

Feeding wheat bran- and barley-based diets that are high in fiber (hemicellulose) increases endogenous losses of Thr in growing pigs compared with those fed a casein-based diet (Myrie et al., 2008). With increasing dietary pectin (soluble fiber), protein deposition was reduced to a larger extent when Thr-limiting diets than Lys-limiting diets were fed, mainly due to increased endogenous loss of Thr (Zhu et al., 2005). More recently, Mathai et al. (2015) reported that the optimum SID Thr:Lys ratio for the average daily gain (ADG) of 25-50 kg pigs fed a high-fiber diet (containing 15% soy hulls) was higher (71%) than that of pigs fed a low-fiber diet (66%). The N retention of pigs fed a high-fiber diet was lower than that of pigs fed the low-fiber diets, indicating that feeding pigs diets high in fiber increases the Thr necessary for mucin production relative to body growth.

Glutamine

Glutamine is the most abundant free AA in the body and milk of mammals (Wu et al., 1996). Besides serving as a major fuel for rapidly dividing cells such as enterocytes and leukocytes of the small intestine, Gln is involved in many metabolic processes, including gluconeogenesis, inter-organ nitrogen transfer, immune response, and regulation of the cellular redox state (Wu et al., 2007). As there is extensive interconversion of Gln and Glu, Glu can partially substitute for Gln in several pathways, including ATP production and the syntheses of Arg, ornithine, citrulline, alanine, proline, and aspartate (Reeds et al., 1997; Wu, 1998). As a precursor of Glu, Gln plays a role in the synthesis of glutathione (Reeds et al., 1997). Glutamine is also a precursor for the synthesis of nucleotides (purine and pyrimidine) that are essential for the proliferation of lymphocytes and mucosal cells (Wu, 1998). The small intestine uses up approximately 70% of ingested Gln in the first pass, and only 30% of Gln in the lumen enters the portal blood pool (Stoll and Burrin, 2006), highlighting the important role Gln plays in maintaining intestinal barrier integrity and immune function.

Table 3 Effect of dietary glutamine on the performance of weaned pigs (Zou et al., 2006)

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A relatively high supplementation with L-Gln at 4% increased white blood cell count and enhanced lymphocyte function in early-weaned pigs infected with E. coli (Yoo et al., 1997). In weaned piglets, supplementing a diet adequate in all AAs with 1% L-Gln increased villus height in the jejunum and increased the gain:feed (Wu et al., 1996). Liu et al. (2002) also reported that supplementing a nutrient-adequate diet with 1% L-Gln or L-Glu increased the jejunal villus height of weanling pigs. Increased BW gain and improved feed:gain of weaned piglets was also observed when 1% L-Gln was included in a diet adequate in all AAs (de Abreu et al., 2010). Zou et al. (2006) reported that supplementation with 1% L-Gln reduced diarrhea incidence and improved growth performance of weaned pigs (Table 3). Similarly, 1% L-Gln supplementation reduced intestinal expressions of genes that promote oxidative stress and increased the intestinal glutathione concentration, small intestine growth, and BW gain of weaned piglets (Wang et al., 2008; Table 4). Dietary addition with a 0.8% 50:50 mix of Gln-Glu increased villus height of the small intestine and the BW gain of weaned pigs (Molino et al., 2012).

Table 4 Effect of dietary glutamine on performance and carcass of weaned pigs (d 21-28; Wang et al., 2008).

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Arginine

Arginine serves as a precursor for the synthesis of important molecules, including nitric oxide (NO), ornithine, citrulline, proline, Glu, creatine, and polyamines (Wu and Morris 1998). Arginine plays an important role in immunity by regulating NO synthesis through nitric-oxide synthase (NOS₂) to produce antibodies through B cells, as well as T-cell receptor expression (De Jonge et al. 2002). Arginine may function as an antioxidant and ameliorate lipid peroxidation (Galli 2007), and also plays a role in the detoxification of ammonia via the urea cycle (Visek, 1986). Moreover, Arg regulates signaling via the mammalian target of rapamycin (mTOR) in the small intestine and skeletal muscle of piglets to initiate body protein synthesis (Yao et al., 2008).

Table 5 Effect of dietary Arg and Gln supply on performance of weaned pigs (Shan et al., 2012).

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Dietary L-Arg addition enhanced the immune status and gain:feed of piglets (Tan et al., 2009a), as well as the immune status of gestating sows (Kim et al., 2006). L-Arginine addition (0.5%) to a diet adequate in all AAs increased serum concentrations of the albumin cytokines IL-2 and IFN-γ and improved the performance of immune-challenged pigs (Han et al., 2009). Supplementation with 1% alleviated the impairment of gut function induced by LPS challenge in weaned pigs (Liu et al., 2009). An L-Arg addition (0.6%) improved the intestinal integrity and growth of weaned pigs (Wu et al., 2010). Improved gut health based on the diarrhea incidence and BW gain of weaned pigs was observed after supplementation with 0.7% L-Arg.HCl or in combination with 1% L-Gln to a diet adequate in AAs (Shan et al., 2012; Table 5).

Table 6 Effect of dietary Arg supply on performance and carcass of 41-90 kg pigs (Tan et al., 2009b).

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These results indicate that Arg supplementation can improve intestinal barrier function. Furthermore, dietary Arg supplementation (1%) increases muscle gain and reduces body fat and plasma triglyceride levels in 41-90 kg pigs (Tan et al., 2009b; Table 6). Supplementation with 1% L-Arg enhanced the anti-oxidative capacity and carcass quality of pigs (Ma et al., 2010).

Glycine

Glycine is involved in the synthesis of many important molecules, including serine, glutathione, creatine, purine nucleotides, and heme (Kim et al., 2007). Indeed, serving as a substrate to synthesize GSH through the small intestinal mucosa is a physiologically important pathway of Gly (Reeds et al., 1997). Glycine plays a role in regulating the production of cytokines through leucocytes and immune function (Zhong et al., 2003). In addition, Gly itself is a potent antioxidant (Fang et al., 2002). In an in-vitro study, adding Gly to the jejunal enterocytes of weaned pigs enhanced cell growth and protein synthesis and reduced attenuated apoptosis when exposed to an oxidative stress model induced by 4-hydroxynonenal (Wang et al., 2014).

Table 7 Effect of dietary supplementations with various amino acids on performance of 20-50 kg pigs (d 0-28; Powell et al., 2011)

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Glycine inhibits glutamine synthase, thereby making more Glu available for the biosynthesis of NEAA (Tate and Meister, 1971). Supplementing Gly or a combination of Gly and Arg (equal to a high-protein diet) to a low-protein corn-SBM diet containing additional Lys, Met, Thr, Trp, Ile, and Val restored growth performance to levels similar to those of 20-50 kg pigs fed a positive control diet (Powell et al., 2011; Table 7), indicating that Gly is an important AA for body protein synthesis.

In conclusion, AAs are involved in important metabolic pathways beyond growth, including modulating the functioning of the body’s immune system. This means that some key AAs such as Met+Cys, Trp, and Thr are prioritized to form compounds involved in the immune response, resulting in compromised growth. This redirection of key AAs implies that dietary supply of these AAs should be increased to maintain optimal immune status and to reduce the negative impact on animal performance. Furthermore, increased dietary supply of Thr, Arg, Gln, and Gly may be beneficial to enhance the immune status and gut integrity of pigs undergoing sub-clinical gut health challenges. Future research is warranted to quantitatively estimate the increased need as well as the interaction or synergetic effects of these functional AAs to improve the immune status, gut health, and performance of pigs raised under sub-optimal conditions.

Acronyms

AAs amino acids

ADG average daily gain

AGPs antimicrobial growth promoters

APP acute phase proteins

Arg arginine

BW body weight

Cys cysteine

EU European Union

FCR feed conversion ratio

FSR fractional synthesis rate

GIT gastrointestinal tract

Gln glutamine

Glu glutamate

Gly glycine

IL interleukin

ISS immune system stimulation

LPS lipopolysaccharide

Met methionine

mTor mammalian target of rapamycin

N nitrogen

NO nitric oxide

PDV portal-drained viscera

ROS reactive oxygen species

S sulfur

SAA sulfur amino acids

SID standardized ileal digestible

Thr threonine

TNF tumor necrosis factor

Trp tryptophan

Roles of functional amino acids in the immune system of pigs

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