Improving protein utilization efficiency through better understanding of immune and stress responses in pigs
Efficient utilization of dietary protein is affected by many factors including the animal’s physiological requirements for amino acids, genetic potential for protein deposition, digestion/absorption of dietary amino acids, use of antibiotics, and metabolism/partitioning of absorbed amino acids. Although other factors are equally important for protein utilization efficiency, this presentation will highlight the roles of immune and stress responses of pigs on protein utilization efficiency as pigs in commercial production system are exposed to considerable environmental stressors that alter the way pigs partition the absorbed nutrients. Emphasis will be given to the currently available nutritional strategies to achieve more efficient protein utilization under such environmental stress.
Retention of dietary protein to body protein is an inefficient process in pigs. Nitrogen balance studies have shown that nitrogen retention rate (proportion of intake) ranges from 41 - 68% in weaner pigs (Cera et al., 1988), 42 - 56% in grower pigs (Fabian et al., 2004, Otto et al., 2003, Zervas and Zijlstra, 2002), 30 - 46% in finisher pigs (Fabian et al., 2004), and 42 - 52% in sows (Renteria-Flores et al., 2008). These findings, based on the mean values from these studies, indicate that approximately 44%, 50%, 64% and 54% of the fed dietary protein is not available for body protein deposition for these classes of pigs, respectively. There are many factors involved in the efficiency of nitrogen retention including: (1) protein intake and body weight (Carr et al., 1977), (2) physiological status such as pregnancy and lactation, (3) obligatory losses during nitrogen turnover in the visceral organs, (4) endogenous losses during digestion and absorption, and (5) dietary factors that reduce N retention efficiency (Dourmad and Jondreville, 2006). Dietary factors affecting nitrogen retention efficiency include dietary energy and amino acid balance (Campbell and Dunkin, 1983), type and amount of dietary fibre (Libao-Mercado et al., 2006, Zervas and Zijlstra, 2002), vitamin and trace mineral deficiency (Miller and Payne 1964), variation in available amino acid content in protein sources which hinder precise formulation and hence maximum efficiency of utilization, unavailability of dietary lysine due to changes in chemical structure mediated during heat processing (van Barneveld et al., 1994) and storage (Rutherfurd and Moughan, 2007), glycaemic index of starch (Drew et al., 2012), nutrient asynchrony (van den Borne et al., 2007, 2012), and frequency of meal (Le Naou et al., 2014). Physiological and dietary factors affecting nitrogen retention in pigs are well documented elsewhere, whilst immunological and neurophysiological factors reducing nitrogen retention efficiency are documented less commonly. Therefore, this paper will attempt to collate available information on the immunological and neurophysiological factors affecting the protein retention efficiency in the commercial pig production system.
Digested and absorbed amino acids across the intestinal epithelium are partitioned to various tissues for either maintenance or body weight gain. As illustrated in Figure 1 (Dourmad and Jondreville, 2006), a large proportion of absorbed amino acids is oxidated and excreted via urine, and the efficiency of N utilization is therefore closely associated with this process. Especially, additional requirements for specific functional amino acids to respond to the immunological and neurophysiological challenges frequently occurring in commercial production systems can alter the ideal amino acid balance and therefore increase amino acid oxidation that becomes excessive under those metabolic conditions.
Figure 1. Nitrogen utilization efficiency in finisher pigs (re-drawn from Dourmad and Jondreville, 2006)
Impact of immune response on protein utilization efficiency
Pigs reared in a commercial production system are exposed to various inflammatory challenges via inhalation or ingestion of antigens and pathogens. Upon the challenge, the innate and adaptive immune system is initiated depending on the nature and the severity of the challenge, and protein retention is reduced due to diversion of nutrients to lymphoid tissues (Klasing, 2004). Interestingly, however, there is a mandatory requirement of energy and amino acids without inflammation for the surveillance system within the gut-associated lymphoid tissue (GALT) when pigs are exposed to the environment that contains antigens/pathogens (Kim et al., 2013). In this regard, microfold cells, dendritic cells and CD4+ in the Payer’s patch and lamina propria are consistently monitoring the presence of antigens/pathogens in the intestinal lumen and pass the information to the lymphoid tissues for preparation of the immune system for possible development of inflammation (Mowat, 2003). Therefore, the presence of antigens/pathogens in the intestinal lumen itself causes extra use of protein and energy that would otherwise be used for tissue deposition in good sanitary conditions (Kim et al., 2012b, 2013). In the case of broiler chickens, housing in a commercial facility reduces growth of chicks by 5% compared with the chicks housed in a germ-free environment (Klasing, 2004, Iseri and Klasing, 2013). In pigs, housing in poor sanitary conditions reduced growth rate by 12% due to reduced feed intake (5%) and poorer feed conversion efficiency (7%; Pastorelli et al., 2012a). Based on those reports, the mandatory requirements of nutrients for the surveillance system in the GALT could be estimated to be around 5-7% in monogastric animals.
Inflammation and subsequent systemic immune response requires much more nutrient diversion to the innate immune system. In broiler chickens, an inflammatory response triggered by intravenous injection of E. coli lipopolysaccharides (LPS) increased the amounts of lysine diverted to the immune system by five times compared with the maintenance level, which is equivalent to a deposition of 7.8 g body mass/day/kg body weight (Klasing, 2004). The LPS challenge in broiler chickens reduced the growth rate of chickens by 17%. However, when weight gain loss due to LPS challenge-associated anorexia was accounted for (40% of the loss), the true cost of the immune system activation in this particular study was around 10.5%. An early pig study which compared protein deposition rates between herds with high and low pathogenic antibody titers demonstrated that pigs with chronic immune system activation (i.e., sourced from a herd with pathogenic antibody titers) showed reduced body protein deposition by 26 - 28% and whole body nitrogen retention rate by 20% (Williams et al., 1997a, b, c), compared to pigs with low pathogenic antibody titers. Similarly, experimental infection with Lawsonia intracellularis decreased feed efficiency by 21% and growth rate by 37% (Paradis et al., 2012). In grower pigs, experimentally-induced chronic immune system activation via repeated intramuscular injections of LPS reduced body protein deposition rate by 12%, while the challenge increased plasma urea nitrogen content by 12%, compared with pigs injected with saline (Kim et al., 2012a). Pastorelli et al. (2012b) conducted a meta-analysis from 122 publications that reported performance of pigs with inflammatory challenges. This particular analysis reported that pigs infected with digestive pathogens, mycotoxicosis, parasites, and respiratory disease challenge reduced growth rate by 40%, 30%, 8% and 25%, respectively. In a commercial finisher pig study, it was reported that concentrations of acute-phase proteins (i.e., haptoglobin and C-reactive protein) in oral fluid samples were negatively correlated to the live weight gain and average daily gain (P<0.001, Seddon et al., 2001).
Such a reduction in nitrogen retention in immune system-activated pigs is due to increased nitrogen oxidation associated with the immune response. The extent of how the nitrogen oxidation would be different depends on the nature and extent of the challenge that triggered the immune response (Pastorelli et al., 2012b, Kim et al., 2013). For example, at the acute phase of the innate immune system activation, absorbed and circulating amino acids and stored globulins in the liver will initially be used for production of acute-phase proteins (Iseri and Klasing, 2013). If circulating or stored amino acids aren’t sufficient for production of related immune molecules then required amino acids would be drained from the skeletal muscle through catabolism. In this respect, an issue of concern is that the ideal amino acid balance required for production of acute phase proteins and immune molecules is different from the ideal amino acid balance obtained from the dietary origin or the skeletal muscle (Rakhshadeh and de Lange, 2011, Kim et al., 2013). Therefore, amino acids that are not used for an immune response and became unbalanced/excessive after body protein anabolism are increasing, which represents the amounts of the increased amino acid oxidized in those situations.
Impact of stress response on protein utilization efficiency
Pigs are exposed to numerous environmental, socio-physical and inflammatory stressors. Exposure to such stressors generally results in a reduction of protein deposition and growth. The endocrinology of the stress response is well documented (Charmandari et al., 2005). Exposure to stress activates the hypothalamic-pituitary-adrenal axis (HPA axis) that suppresses growth hormone secretion and release of glucocorticoids, which in turn inhibits the effects of insulin-like growth factor-1 (IGF-1) and other growth factors in the target tissues. Corticotropin-releasing hormone (CRH) (or corticotropin-releasing factor; CRF) is the major hypothalamic regulator of the pituitary-adrenal gland. Corticotropin-releasing hormone stimulates release of adrenocorticotropic hormone (ACTH) in the pituitary gland, which signals the adrenal gland to release glucocorticoids such as cortisol (Charmandari et al., 2005). Stress-induced stimulation of HPA axis and resultant glucocorticoid release are known to increase visceral adiposity and decrease muscle and bone mass (Charmandari et al., 2005).
In pigs, Moeser et al. (2007) demonstrated that early weaning stress (earlier than 21 days) increased serum CRF and cortisol concentrations and decreased intestinal barrier function (i.e., increased transcellular and paracellular permeability). Mucosal CRF concentration and expression of CRF receptors in the jejunal epithelium were also increased by early weaning stress (Moeser et al., 2007). Later, using an Ussing-chamber model, Overman et al. (2012) demonstrated that exposure to CRF in the ileal tissue increases release of the pro-inflammatory cytokine TNF-α and stimulates mast cell degranulation, which are important sentinel cells for an immune response. The study also demonstrated that exposure to CRF increased paracellular permeability in the ileum. A subsequent study from the same research group demonstrated that early weaning stress increased the risk of inflammation when experimentally challenged with F18 enterotoxigenic E. coli (ETEC) compared with pigs that had reduced stress, by delaying the weaning age (McLamb et al., 2013). Growth rate in pigs with early weaning stress (16-18 days) was significantly decreased when they were challenged with ETEC compared to the non-infected control group, while ETEC infection only marginally reduced growth rate in piglets with delayed weaning to 20 days of age. This result indicates an important interaction between stress and susceptibility to inflammation, which is mediated by stimulation of HPA axis and release of CRF.
Exposure to heat stress is also known to rapidly degenerate intestinal integrity, increasing circulating endotoxin, intramuscular oxidative stress and TNF-α abundance in pigs after short-term exposure (24-72 hours) to heat stress. Epithelial barrier function is subsequently compromised (Montilla et al., 2014, Pearce et al., 2014, 2015). In addition, exposure to heat stress for 12 hours increased NF-кB signaling in oxidative skeletal muscle of growing/finishing pigs, indicating heat stress-associated activation of inflammatory signaling (Genesan et al., 2016).
Nutritional strategies to minimize the impacts of immune and stress responses on protein utilization efficiency
Tryptophan and sulphur amino acids
Response to inflammatory challenge initiates with production of acute-phase proteins, and most acute-phase proteins contain higher tryptophan (Trp) and sulphur amino acids levels (SAA, Reeds, 1994). Moreover, interferon gamma (IFN-γ) produced by immune cells stimulates expression of indoleamine 2,3 dioxygenase (IDO) in the liver, which facilitates conversion of Trp to L-kynurenine. Kynurenine then further is converted to 3-hydroxykynurine and 3-hydroxyanthranilate that scavenge free radicals produced during the immune response (Le Floc’h and Seve, 2007). Due to increased catabolism of Trp to kynurinine, pigs with an inflammatory response showed decreased plasma Trp content (Le Floc’h and Seve, 2007) and therefore reduced growth and deteriorated feed efficiency (Floc’h et al., 2004). Using a chronic immune system stimulation model in 20 kg pigs, De Ridder et al. (2012) demonstrated that (1) immune system stimulation increased urinary nitrogen excretion in the acute phase (3 days after LPS challenge) but not in the adaptive phase (7 days after infection), and (2) increasing Trp intake linearly improved whole body protein deposition. The authors estimated that LPS challenge increased Trp requirement for whole body protein deposition by 7%. In an ETEC challenge model, Trevisi et al. (2009) demonstrated that the inflammatory challenge decreased the average daily gain in ETEC-susceptible pigs, and increasing the Trp to lysine ratio (Trp:Lys) from 0.18 to 0.22 improved average daily gain for only the first 4 days after the challenge. A similar short-term positive effect of Trp supplementation in ETEC-challenged pigs was demonstrated by a recent study (Capozzalo et al., 2016). Increasing the standardized ileal digestible (SID) tryptophan to lysine ratio (Trp:Lys) from 0.16 to 0.24 suppressed the inflammatory response as measured by the acute-phase protein index (Figure 2) at 24 hours after the ETEC challenge, but did not suppress inflammatory response at 7 days after the ETEC challenge (Capozzalo et al., 2016). As a response, pigs supplemented with increased Trp improved their feed conversion ratio in the first 2 weeks after weaning (Capozzalo et al., 2016, Figure 3). Collectively, these studies indicate that inflammatory challenges increase Trp metabolism through the kynurenine pathway, and supplementation of Trp reduces the acute phase inflammatory response and improves protein utilization efficiency.
Figure 2. Acute phase protein index [(C-reactive protein x PigMAP)/ApoA1] in weaner pigs experimentally challenged with an enterotoxigenic strain of E. coli (ETEC) and fed diets supplemented with additional tryptophan and sulphur amino acids (above the NRC 2012 recommendation). Pigs were challenged with ETEC on days 5, 6 and 7, and blood samples were collected before infection on day 5, 24 hours after infection on day 8 and on day 14 to examine acute and lasting effects of dietary supplements on inflammatory response of pigs (Capozzalo et al., 2016).
It has been demonstrated that the response to an inflammatory challenge (i.e., LPS) upregulates hepatic gene expression for cystathionine β-synthase and cystathionine γ-lyase to increase conversion of methionine to cysteine, which is then used for synthesis of glutathione and taurine (Rakhshandeh et al., 2010a). Increased use of SAA in immune system-challenged pigs was also demonstrated in a nitrogen balance study where repeated LPS challenge in grower pigs increased sulphur retention while nitrogen retention was decreased (Rakhshandeh et al., 2010b). This particular study indicates that when there is an inflammatory challenge SAA can limit efficient utilization of the other amino acids. In grower/finisher pigs, Kim et al. (2012a) demonstrated that (1) chronic immune system activation simulated by repeated intramuscular injection of LPS reduced body protein deposition rate by 12%, and (2) increasing SAA supplementation by 20% over the current NRC (2012) recommendation improved body protein deposition rate similar to that of the sham-infected pigs. A dose-response study with weaner pigs reported optimum SID SAA:Lys ratios of 0.71 and 0.68 for maximum daily gain and feed utilization efficiency, respectively, when pigs were orally challenged with ETEC (Capozzalo, 2015). These levels of dietary SAA are 29% and 23% higher levels than the current recommended level of SID SAA:Lys 0.55 (NRC, 2012). A subsequent study demonstrated that increasing SAA supplementation to 60% of Lys in ETEC-challenged weaner pigs significantly suppressed the inflammatory response measured as the acute-phase index up to 7 days after infection (Figure 2), and significantly improved feed efficiency (Figure 3, Capozzalo et al., 2016). Also an interesting finding in this particular study was that increasing SAA and Trp in combination further suppressed acute-phase proteins and tended to further improve feed efficiency compared to individual supplementation of either SAA or Trp (Figures 2 and 3).
Figure 3. Feed conversion ratio in weaner pigs experimentally challenged with an enterotoxigenic strain of E. coli (ETEC) and fed diets supplemented with additional tryptophan and sulphur amino acids (above the NRC 2012 Recommendation). Pigs were challenged with ETEC on days 5, 6 and 7, and FCR was measured over 14 days after weaning. Effect of SAA P<0.05, Effect of Trp P<0.05, SAA x Trp interaction P=0.092 (Capozzalo et al., 2016).
Tryptophan to large neutral amino acids ratio (trp:lnaa ratio)
Tryptophan is a precursor for the synthesis of serotonin that inhibits physiological stress by reducing response of the HPA axis and glucocorticoids (Le Floc’h et al., 2007). Diets low in Trp impair serotonin production in the brain, resulting in depressed feed intake and increased stress response (Markus, 2008). Using a mixing stress model in weaner pigs, Koopmans et al. (2006) demonstrated that supplementation of 5 g Trp/kg diet increased hypothalamic serotonin turnover and reduced plasma cortisol and noradrenaline concentrations after exposure to 3 days of mixing stress. When 12 kg weaned pigs were exposed to repeated isolation and mixing stress, increasing Trp intake linearly decreased the plasma urea nitrogen content while linearly increasing average daily gain up to daily Trp intake reached to 6.6 g/day and 10.8 g/day, respectively, which are very high levels considering current recommended dietary level of Trp of 2.5 g/kg diet (Shen et al., 2012a).
The availability of Trp for synthesis of hypothalamic serotonin is affected by amounts of free and protein-bound Trp in the diet and the Trp to large neutral amino acids ratio (LNAA, isoleucine, leucine, valine, phenylalanine, and tyrosine), as LNAA compete with Trp for passage through the blood-brain barrier (Koopmans et al., 2005). Shen et al. (2012b) imposed social mixing stress to 6-week-old pigs and compared diets with either 0.2 vs. 0.79% Trp in combination with a LNAA concentration of either 4.5 or 3.8% in a 2×2 factorial arrangement. Supplementing Trp significantly increased hypothalamic serotonin turnover and reducing LNAA significantly decreased salivary cortisol concentration after exposure to the mixing stress. Increasing Trp and reducing LNAA concentration in the diet additively reduced plasma urea concentration and also additively improved feed utilization efficiency. Similar studies repeated in a commercial piggery consistently showed that increasing SID Trp to 0.98%, and reducing the LNAA concentration from 4.5 to 3.8% in a diet for weaner pigs, decreased salivary cortisol content and plasma urea nitrogen, and improved daily gain and feed utilization efficiency, especially immediately after mixing (Shen et al., 2015). Collectively, these studies demonstrated that supplementing a larger amount of Trp and reducing the LNAA concentration increased hypothalamic serotonin turnover and reduced the release of glucocorticoids, which in turn reduced stress-associated deterioration of protein utilization efficiency.
Immune and stress modulators
One of the primary responses after exposure to inflammatory and environmental stresses is the release of hypothalamic CRF and activation of mast cells, which orchestrates the immune response (Kim et al., 2013). Overman et al. (2012) demonstrated that early weaning stress increased mucosal CRF concentration that stimulated mast cell degranulation, which deteriorated intestinal integrity. However, when the ileal epithelium of weaned pigs was exposed to a mast cell stabilizer sodium-cromolyn, CRF-induced mast cell degranulation and intestinal barrier dysfunction were reduced. A recent study examined effects of cromolyn on weaning stress-induced activation of CFR-mast cell axis, and reported that intraperitoneal injection of sodium cromolyn (20 mg/kg body weight) at -0.5, 8 and 16 hours relative to weaning improved intestinal barrier function and improved daily gain and feed utilization efficiency by 19% and 50%, respectively (Mereu et al., 2015). On the other hand, antagonising glucorcorticoids can be another way to reduce immune and stress response. Wooten et al. (2016) intramuscularly injected the cortisol agonist dexamethasone (0.2 mg/kg body weight) 1 day before and 3 days after weaning in segregated early-weaned piglets. The cortisol agonist reduced plasma haptoglobin and IL-1β by 1.9-fold and 2.9-fold, respectively, and improved feed utilization efficiency by 60%. These studies indicate that the use of either mast cell stabilizer or cortisol agonist can attenuate immune- and stress-induced reduction in protein utilization efficiency.
Supplementation of antioxidants and acetylsalicylic acid can reduce the negative impact of immune and stress responses by improving whole body antioxidant capacity and reducing acute-phase inflammatory response. A recent study demonstrated that supplementation of vitamin E at 200 IU/kg diet reduced the plasma haptoglobin content immediately after oral ETEC challenge in weaned pigs (Kim et al., 2014). Also, supplementation of 125 ppm acetylsalicylic acid in ETEC-challenged weaner pigs significantly reduced plasma urea concentration and prostaglandin E2 in the liver and tended to improve feed utilization efficiency (Kim et al., 2014).
Exposure to inflammatory challenge and environmental stressors in commercially reared pigs are important contributors to the efficiency of protein retention. Diversion of energy and amino acids to address inflammatory and environmental challenges result in a reduction in feed utilization efficiency by 10 - 40% depending on the type and severity of the challenge. Supplementation of specific functional amino acids or manipulating ratios between amino acids that are extensively involved in immune and stress response is suggested as strategies to reduce nutrient diversion into immune and stress response systems. Moreover, manipulating the cellular signaling mechanisms via use of a mast cell stabilizer and glucocorticoid agonist warrants further investigation.