The gut microbiota play a critical role in regulating and controlling of host energy metabolism (Cani and Delzenne, 2009; Yan et al., 2012). The hindgut of an animal harbors a dense and metabolically active microbiota comprising primarily bacteria that have a profound influence on immunological, nutritional, and physiological processes in the host (Su et al., 2008). Various factors can influence on the composition of the gut microbiota, such as nutritional and non-nutritional dietary components, the intestinal environment, and antibiotic use (Penders et al., 2006). In mammals, proteins and their amino acid-derived metabolites can affect the interactions between the gut microbiota and metabolism (Metges, 2000). Undigested protein in the large intestine can be fermented by indigenous bacteria to form amino acid-derived metabolites (Nyangale et al., 2015; Zhang et al., 2016), such as ammonia (NH₃), branched chain fatty acids (BCFAs), hydrogen sulfide, and indolic and phenolic compounds, all of which can possibly have toxic impacts on the intestinal epithelium (Geypens et al., 1997; Luo et al., 2015). In pigs, the level of protein in the diet affect the composition and metabolism of bacteria, furthermore, the crystalline amino acids (AAs) -supplemented low-protein (LP) diet may decrease the formation of protein fermentation products, and alter microbial communities in the large intestine (Zhou et al., 2015; Zhang et al., 2016). Moreover, the addition of 3% benzoic acid to a low protein diet can reduce the NH₃ concentration and NH₃ emissions by 55 and 57%, respectively (Hansen et al., 2007). Benzoic acid also diminishes the growth of micro-organisms in the intestinal digesta of piglets (Kluge et al., 2006). Therefore, it is important that both researchers and swine managers understand how dietary interventions can lead to differential effects on microbial composition and fermentation.

Alpha-ketoglutarate (AKG) is a central molecule in the tricarboxylic acid cycle (Krebs cycle). It can be rapidly transaminated, by glutamate (Glu) dehydrogenase, to glutamic acid and then further aminated, by glutamine (Gln) synthetase, to form Gln (Prandini et al., 2012; Wang et al., 2016). This conversion is well-established (Wang et al., 2015):

In addition to functioning as both an energy donor and a scavenger of ammonium ions, AKG can provide a source of Gln that stimulates protein synthesis in intestinal epithelial cells (Yao et al., 2012). Furthermore, Junghans et al. (2006) reported that administration of AKG may markedly affect energy expenditure and nutrient utilization in growing pigs. Dietary supplementation with 1% AKG beneficially modulates the AMPK signaling pathway to improve energy status in the small intestine of LPS-challenged piglets (Hou et al., 2011b). Daily weight gain and feed-efficiency was improved when pigs were fed supplemented with AKG (Hou et al., 2011a; Chen et al., 2017a). In addition, the in vitro bacterial community structure is changed when AKG is introduced into diluted rumen fluid (Zhang et al., 2011). We have previously shown that dietary supplementation with AKG can potentially promote the growth of beneficial bacteria, improve intestinal microbial populations, modulate the production of short-chain fatty acids (SCFAs), and decrease the level of NH₃ in the gut of growing pigs (Chen et al., 2017b).

Our study objective was to investigate how combining a LP diet with AKG might influence growth performance, cecal microbial communities, and parameters of microbial metabolism in young pigs. The data obtained here will help us further understand the mechanism through which AKG improves pig performance, and provide a scientific basis for using AKG in commercial practice.

These experiments were conducted in accordance with Chinese guidelines for animal welfare and experimental protocols, and all procedures were approved by the Committee of Animal Care at the Institute of Subtropical Agriculture, Chinese Academy of Sciences. The AKG component (purity ≥ 98%) was obtained from Hubei Yuancheng Saichuang Technology Co., Ltd., Wuhan 430064, China.

Organic acids and their salts are considered a class of natural growth promoters that have long been used as feed additives in pig nutrition programs to reduce the buffering capacity of starter diets (Giannenas et al., 2016). Various organic acids (e.g., formic, citric, fumaric, malic, and benzoic) can improve growth performance and regulate the intestinal microbiota in pigs (Partanen and Mroz, 1999; Piva et al., 2002; Kluge et al., 2006; Øverland et al., 2008; Halas et al., 2010). However, the strategy of using a low protein diet balanced with AAs has no influence on growth performance in commercial swine operations (Opapeju et al., 2008) and broiler chickens (Kobayashi et al., 2013). Our research goal was to examine how, while applying current production practices, a low protein diet (3% below normal) could be supplemented with AKG to improve growth performance, the microbial communities, and metabolic profiles in the cecum of a growing pig. In fact, the LP diet alone had no impact on the values calculated for ADG, ADFI, or F/G. This outcome is consistent with that reported by Le Bellego and Noblet (2002), who showed that reducing the protein level from 204 to 169 g/kg did not affect feed intake nor daily weight gain of the piglets (12 kg initial BW). However, we did note here that the ADG was increased by 7.23% in pigs that received an LP diet supplemented with AKG when compared with the results from the LP group. This is in accord with findings from previous studies (Hou et al., 2011a; Chen et al., 2017a). It is possible that the dietary AKG supplementation promotes intestinal development (Hou et al., 2010), and increases AA- and energy-digestibility and nitrogen deposition (Chen et al., 2017a), while reducing the populations of harmful bacteria and modifying their metabolites within the gastrointestinal tract (Chen et al., 2017b). Furthermore, AKG is an intermediate of the citric acid cycle and the natural ubiquitous collector of amino groups in body tissues. It has been reported that AKG can stimulates AMPK phosphorylation and oxidation of energy substrates in the intestinal mucosa (Hou et al., 2011b), thereby inactivating one of its downstream target enzymes, acetyl-CoA carboxylase (ACC) (Dyck et al., 1999), which converts acetyl-CoA into malonyl-CoA (an inhibitor of fatty acid oxidation and a substrate for fatty acid synthesis) (Hou et al., 2011b) and enhancing ATP supply, thereby improving fat storage and weight gain. This was reflected by the rise in ADG in those pigs fed ALP diets over the entire experimental period.

Because the gut microbial community has potentially important roles in swine growth performance and health, its composition and metabolic activities in various nutritional and physiological contexts deserve close attention (Hill et al., 2005). It has been demonstrated that the composition of the intestinal and fecal microbiota (including the bacterial profiles of the mucosa and digesta) changed in pigs from birth to the finishing phases (Konstantinov et al., 2006; Kim et al., 2011). We comprehensively evaluated the diversity in that microbial community as well as the composition of cecal samples from growing pigs by using 16S rRNA gene sequencing. On average, 38,384 effective reads were obtained for each sample (Supplementary Table S1), with high coverage (>99.07%) (Supplementary Table S2). Furthermore, the microbial richness and diversity indices in the cecum were not influenced by diet (Supplementary Figure S1 and Supplementary Table S2). At the phylum level, the cecal microbial communities in growing pigs were dominated by Firmicutes (41.69–56.57% of the total microbial content), Bacteroidetes (32.25–35.12%), and Proteobacteria (7.11–14.04%) (Figure 3). This observation is in accordance with the findings of Ji et al. (2017) and Isaacson and Kim (2012). We also found that, at the genus level, the AKG-supplemented LP diet increased the abundances of Lachnospiraceae, and Phascolarctobacterium species from the Firmicutes phylum, and Parabacteroides species from the Bacteroidetes phylum (Figures 4, 5). Some bacteria belonging to the Firmicutes phylum can efficiently ferment fiber into butyrate, while some bacteria from the Bacteroidetes phylum ferment fiber into acetate and propionates (Zhou et al., 2017). Moreover, microbial community structure in piglets fed with ALP was remarkably different from that in LP group or NP (Figure 6A). Furthermore, the HCA showed that the similarity dendrogram was separated by AKG rather than protein level (Figure 6B). This suggests that the change in gut microbiota structure results from its accommodation to an intestinal environment created by adding AKG to the diet. As described below, there were some changes in intestinal microbial composition, and consequently altered metabolites types and concentrations of cecal digesta under ALP diet.

Bacteroides and Bifidobacterium are the two main microbial genera that most significantly affect energy homeostasis and substrate fermentation in the hind gut (Tennyson and Friedman, 2008). In pigs, Bacteroides species may compose a small fraction of the phylum Bacteroidetes community while other members of this phylum may bound up with the development of obesity (Yan et al., 2012). We found that the LP diet increased the amounts of cecal Bacteroides (Figure 7), an outcome consistent with that from a previous study (Zhang et al., 2016). The ALP diet also increased the amounts of cecal Bacteroides and Bifidobacterium (Figure 7), perhaps leading to AKG-induced fat accumulations. This was reflected by the rise in ADG in those pigs fed ALP diets over the entire experimental period. These findings partly agree with those described previously. For example, Chen et al. (2017b) demonstrated that supplementing the dietary feed with AKG elevated the cecal Bifidobacterium population in growing pigs. Our investigation also revealed that a LP diet supplemented with AKG increased the level of cecal Lactobacillus and decreased the E. coli population when compared with the other diet groups. These microorganisms – Lactobacillus and E. coli – are generally considered to be beneficial and harmful, respectively, to the host animal, and Lactobacillus is a commonly used probiotic and is thought to create a barrier against pathogen colonization (e.g., by coliform bacteria) in the proximal part of the alimentary tract of young animals (Knarreborg et al., 2002). Because AKG is a weak acid (Filip and Pierzynowski, 2007; Liem et al., 2008), it can only diminish the effect of intestinal pathogenic bacteria (such as coliforms), but not effectively inhibit the activity of useful bacteria, including Lactobacillus, largely because of the pH-insusceptibility of that organism (Kazempour and Jahanian, 2017). Furthermore, Glu and several other amino acids are primarily utilized by the bacteria in the intestines of pigs, where they play crucial roles in preserving intestinal functions and health, including the balance of microflora (Dai et al., 2010, 2012).

Profiles of microbial metabolites can be used to represent microbial activity and intestinal health. Some beneficial bacteria (e.g., Bacteroides and Bifidobacterium) can ferment carbohydrates to produce high concentrations of SCFAs (e.g., acetic acid, butyric acid, valeric acid, and propionic acid), which in turn can decrease the pH of their environment and inhibit the growth of other bacteria (Arunachalam, 1999; Olukosi and Dono, 2014; Chen et al., 2017b). Generally, valeric acids and branched-chain (isobutyric and isovaleric acids) are considered as the end products of protein fermentation (Williams et al., 2015). Protein levels in dietary feed can affect microbial fermentation in the cecum (Zhang et al., 2016). When compared with the NP diet, the LP diet can decrease the amounts of isobutyrate and isovalerate in the cecum (Zhou et al., 2015). Similarly, we found that the LP diet decreased the isobutyrate concentration (Figure 8). Furthermore, supplementing this LP diet with AKG increased the concentrations of valerate and propionate (Figure 8). We have previously reported that the dietary AKG supplementation enhances the concentrations of butyric acid and valeric acid in the cecal digesta (Chen et al., 2017b). Halas et al. (2010) also showed that dietary benzoic acid supplementation increases the relative amount of valeric acid in the cecum. In the present study, it’s possible that the increase of valerate and propionate could be well explained by the increase of Bacteroides and Bifidobacterium (Arunachalam, 1999). All of these results suggest that fewer indigestible dietary proteins are entering the cecum and are being degraded by cecal microbial proteases. Energy is the limiting factor for microbial growth in the hindgut, the primary site of intestinal fermentation (Piva et al., 2002). Therefore, AKG can serves as a metabolic fuel for the gut in animals fed an LP diet, thus sparing other oxidative fuels (Lambert et al., 2002). As a valuable source of energy for the intestinal microbiota, AKG improves bacterial fermentation in those parts of the digestive tract, thereby modulating the production of SCFAs and BCFAs.

Ammonia is a toxic catabolite produced through urea hydrolysis and the degradation of microbial amino acids, may interfere with epithelial cell turnover and depress the growth performance of pigs (Walsh et al., 2007). Swine productivity is not necessarily diminished if one can reduce the amount of crude protein and, ultimately, that of ammonia while still maintaining an adequate level of essential amino acids (Canh et al., 1998; Hayes et al., 2004; Hansen et al., 2007). Similarly, we found here that pigs receiving the LP diet showed a decreased ammonia concentration in the cecum (Figure 9). That concentration was also significantly reduced in the cecum when the LP diet was supplemented with AKG. These results are in accord with those reported previously (Hansen et al., 2007; Chen et al., 2017b). The LP diet supplementation with crystalline AAs may deter the formation of protein fermentation products in the large intestine (Luo et al., 2015; Zhou et al., 2015). Moreover, the addition of appropriate exogenous organic acids to pig diets may increase the apparent digestibilities of protein and AAs, while also reducing the production of NH₃ (Partanen and Mroz, 1999; Murphy et al., 2013). All of these findings suggest that deamination and decarboxylation of dietary and/or endogenous protein by microorganisms in the gastrointestinal tract are altered by such supplementation and respond to an LP diet. These responses could be due to a decline in the microbial fermentation. LP diet may decrease amino acid breakdown by cecum bacteria or increased incorporation of amino acids into microbial biomass (Pieper et al., 2012; Zhang et al., 2016). The improvement on amino acids digestion and absorption results in better N digestibility and less NH3 excretion (Chen et al., 2017a). As an intermediate of the citric acid cycle and a natural, ubiquitous collector of amino groups in body tissues, AKG can decrease the level of free NH₃ by reducing glutamate and glutamine oxidation and/or by incorporating NH₃ into glutamate by the reversible enzyme GDH (Wirén et al., 2002; Lambert et al., 2006). In addition, dietary AKG supplementation can increase glutamine synthetase gene expression in intestine of common carp (Cyprinus carpio) (Dong et al., 2014). Therefore, this supplement has a beneficial effect on N metabolism (Wirén et al., 2002) and can decrease the toxicity of ammonium ions in an organism (Stoll et al., 1991; Velvizhi et al., 2002). Our results confirm those previously showing that AKG is able to replace dietary dispensable AAs by shunting ammonium back into the dispensable AA pool (Filip and Pierzynowski, 2007; Chen et al., 2017b). Moreover, it has been suggested that N retention is good in infants with a bifidus microflora, Bifidobacterium promotes the amino acids metabolism. The ability of Bifidobacterium to utilize NH₃ as a source of N may also lead to decrease of NH₃ in the colon (Arunachalam, 1999). Therefore, we speculate that the decreasing of NH₃ could be also associated with increasing proportions of Bifidobacterium in the current study.

We investigated the effects on growth performance, cecal microbial communities, and parameters of microbial metabolism in the cecum when pigs were fed a LP diet supplemented with AKG. In particular, this addition altered microbial communities by increasing of the SCFA-producing bacteria and the amounts of Bacteroides and Bifidobacterium, while also decreasing Escherichia coli and the concentration of ammonia in those tissues, thereby increasing ADG of growing pigs. These findings may help us understand the mechanisms by which AKG improves pig performance, and provide additional information that might support the future use of this supplement in animal production.

JC and KY conceived the experiment and revised the manuscript. JC, QJ, MH, and BK performed the experiments. JC, QJ, CF, LL, and BK provided the reagents and experimental materials. JC analyzed the data and wrote the manuscript. LL, YZ, and CF prepared the figures and edited the manuscript. All authors reviewed the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.