Low-protein diets, namely having a reduction in the crude protein level than the recommended level (NRC, 2012) followed by meeting the amino acid (AA) requirements for pigs, have gradually been applied in swine production due to multiple beneficial effects like reducing feed costs and nitrogen excretion. Previous studies have demonstrated that reducing crude protein levels by 2% to 4%, followed by the appropriate addition of essential AA, is an effective strategy to reduce nitrogen emissions, the risk of gut disorders, and feed costs, without compromising animal performance (Gloaguen et al., 2014; Jiaqi et al., 2020; Wu et al., 2015). It has been generally recognized that non-essential amino acids (NEAA) also play a vital role in achieving maximum growth and production performance, despite more emphasis on essential AA in the past (Wu et al., 2013). Some NEAA have higher utilization rates than synthesis rates in some circumstances and therefore are categorized as conditionally essential AA (Wu, 2009). The conditionally essential AA for weaned piglets are glutamic acid (Glu), glutamine (Gln), arginine, proline, glycine (Gly), and taurine (Upadhaya et al., 2022). With the increased availability of crystalline AA, formulating low-protein diets with a balanced AA content has become possible. However, knowledge regarding the effects of dietary supplementation of conditionally essential AA on growth performance in low-protein diet-fed piglets is limited.

Gln, one of the conditionally essential AA for pigs (Wang et al., 2015a), is the most abundant AA in sows' milk during a certain stage of lactation (22 to 29 d) and is also rich in plasma, body fluid, and skeletal muscle (Wu and Knabe, 1994; Wu et al., 2013). Exhaustion of Gln would give rise to considerable detrimental effects like villus atrophy, downregulation of the abundance of tight junction proteins, and increased paracellular permeability, and this phenomenon could be alleviated by Gln supplementation (Kim and Kim, 2017). Moreover, inadequate intake of Gln is closely related to the breakdown of the intestinal barrier and the development of diseases, which could be improved by Gln supplementation (Bertrand et al., 2016; Chaudhry et al., 2016; Zhou et al., 2010). Of note, Gln has been recommended as a feed supplement to reduce weaning stress in piglets (Ji et al., 2019), and the use of Gln in animals is safe and efficacious according to the European Food Safety Authority (Bampidis et al., 2020). Our previous results demonstrated that dietary 1% L-Gln increased the body weight and improved intestinal histomorphology and barrier function in weaned piglets (Wang et al., 2015b). We also found that L-Gln could prevent high-fat diet-induced metabolic disorders in rats (He et al., 2022), enhance tight junction integrity via Ca²⁺/calmodulin-dependent protein kinase (CaMK)-adenosine monophosphate-activated protein kinase (AMPK) signaling (Wang et al., 2016), and ameliorate 4-hydroxy-2-nonenal-induced apoptosis by modulating glutathione (GSH)-related redox homeostasis (Liu et al., 2018). However, information regarding the effects of dietary Gln in piglets under low-protein diets remains limited.

In recent years, the gut microbiota has attracted the attention of many scientists as it exerts diverse beneficial effects on hosts like the fermentation of carbohydrates, maintenance of intestinal normal functions, protection from pathogenic bacteria, and modulation of immunity (Buffie and Pamer, 2013; Kamada et al., 2013). The swine gut microbiota is a complicated ecosystem exhibiting dynamic composition and diversity that shifts with time and along the entire gastrointestinal tract (Isaacson and Kim, 2012), and is implicated with the physiological status and developmental stage, nutritional composition, pathogen infection, and so on (Ji et al., 2017). Gut microbiota disruption is generally supposed to be one of the key factors resulting in postweaning diarrhea during the weaning transition, and the rehabilitation or reconstruction of the gut microbiota at an early age is critical to the host's health (Gresse et al., 2017). Importantly, recent data have revealed that the AA metabolism can alter the gut microbiota (Heianza et al., 2019). To the best of our knowledge, however, the studies regarding the effect of dietary Gln on gut microbiota in low-protein diet-fed piglets are limited. Given the multiple beneficial effects of Gln, we explored the regulatory effects of different levels of dietary L-Gln on growth performance, serum profile, redox status, and gut microbiota in low-protein diet-fed piglets.

Gln is a conditionally essential AA for pigs and has been recommended as a feed supplement to reduce weaning stress in piglets due to its multiple beneficial effects (Ji et al., 2019). Substantial studies have investigated the effects of dietary Gln on growth in pigs. Yi et al. (2005) found that 2% (as-fed basis) Gln improved ADG and gain to feed ratio in weaned pigs at 48 h after E. coli K88 infection. Moreover, Kitt et al. (2002) found that Gln improved feed efficiency from d 14 to 21 in piglets during the early post-weaning period. Likewise, Wu et al. (1996) reported that 1% of Gln supplementation not only enhanced the gain to feed ratio by 25% but also prevented jejunal atrophy. Weanling pigs fed the 1% L-Gln diet had higher ADG and gain to feed ratio than those fed the control diet during d 0 to 14 and d 1 to 28 (He et al., 2016). In the present study, we advanced the knowledge that a low-protein diet supplemented with 1% L-Gln enhanced growth performance, while 2% and 3% L-Gln dramatically inhibited growth performance in low-protein diet-fed piglets, suggesting that the concentration of L-Gln should be less than 2% in low-protein diet-fed piglets. Our further investigation demonstrated that piglets fed diets containing 2% or 3% of L-Gln couldn't metabolize the extra L-Gln as evidenced by the increased blood urea nitrogen. Besides, supplementation of 2% or 3% L-Gln may also induce the antagonism of other AA like arginine and proline, which, in turn, inhibits growth performance (Holecek, 2013). Of note, a recent study demonstrated that pigs fed diets (normal protein level) with Gln inclusion improved growth performance after weaning when compared with the control, with 0.4% Gln being the most effective level among the treatments (con, 0.2%, 0.4%, 0.6%, 0.8% and 1% of L-Gln) (Duttlinger et al., 2020), indicating the optimum concentration of L-Gln in low-protein diet may be similar. More studies regarding the effects of dietary L-Gln on growth performance in low-protein diet-fed piglets need to be conducted.

Serum physiochemical parameters like AST, ALT, and glucose can reflect the physiological status of the organism to some degree. AST plays a vital role in modulating cell proliferation by participating in AA metabolism, especially Gln metabolism (Song et al., 2022). Briefly, when Gln enters the cytoplasm, it is subsequently hydrolyzed into Glu and ammonia (Wise et al., 2008). The Glu converts into α-ketoglutaric acid and is further catabolized in the tricarboxylic acid (TCA) cycle. On the other hand, Glu can also formulate aspartate by AST 2, then the aspartate from the mitochondrial matrix undergoes AST 1-catalyzed reaction in the cytoplasmic matrix to generate oxaloacetate, and finally converts into pyruvate, during which nicotinamide adenine dinucleotide phosphate (NADPH) is also produced (Li and Le, 2018). Moreover, ALT, also known as glutamic pyruvic transaminase (GPT), is a gluconeogenesis enzyme that catalyzes conversions between alanine and pyruvic acid. ALT, along with AST is often used as a blood biomarker of hepatic damage. In the current study, piglets on d 14 had lower activities of serum AST and ALT in response to 1% L-Gln administration, and our further study found that a low-protein diet supplemented with L-Gln enhanced serum Gln content, whilst reducing serum aspartate and β-alanine levels linearly, indicating that the cellular energy pathway regarding Gln converting into Glu is inactive. The elevation of serum Gln levels may be attributed to an augmented uptake of Gln by the small intestinal epithelium, which subsequently acts as a compensatory mechanism to offset Gln catabolism. Consistently, the serum glucose was observed to be lower in response to 1% L-Gln; these results together suggested that 1% Gln exerted other functional roles beyond supplying energy in low-protein diet-fed piglets at an early age. Intriguingly, previous studies demonstrated that a normal protein diet supplemented with 1% Gln increased ALT activity, with no significant effect on AST (Xiao et al., 2012), which may be related to the protein level in the diet, the age, and the physiological state of piglets.

The antioxidative function is one of the multiple beneficial effects of Gln since Gln can convert into Glu, which is the precursor substance for the synthesis of GSH. GSH is an important regulator of the cellular redox state protecting cells from damage caused by lipid peroxides, reactive oxygen and nitrogen species, and xenobiotics (Kennedy et al., 2020). H₂O₂ is an endogenous reactive oxygen species (ROS) that leads to oxidative stress directly as a molecular oxidant and indirectly through free radical generation (Murphy and Friedman, 2019), which can be hydrolyzed water and oxygen by CAT, and SOD is another important antioxidant enzyme in scavenging ROS. Moreover, the MDA level is the main product of lipid peroxidation by ROS and is therefore deemed a lipid peroxidation biomarker (Placer et al., 1966). He et al. (2016) demonstrated that feeding a 1% L-Gln diet (protein level 20.4%) increased SOD activity while decreasing the serum MDA content on d 28 in weanling pigs. Likewise, higher activities of jejunal mucosa glutathione peroxidase (GSH-Px) and T-SOD were observed in 1% Gln-fed piglets, with the MDA concentration being simultaneously reduced (Zhang et al., 2017a). Consistently, in the present study, a low-protein diet supplemented with 1% L-Gln reduced serum MDA and H₂O₂ levels, and MPO activity, whilst enhancing activities of CAT and T-SOD, and GSH content in pigs on d 14. Of note, there is a negative correlation between serum Gln contents and mucosal barrier function (Wang et al., 2008), suggesting there may be similar relationships between serum Gln or other AA like Gly. Our studies further revealed that the L-Gln administration reduced serum Glu and Gly levels in piglets, which may account for the increase in GSH since both Glu and Gly are precursors of GSH. The inconsistency of antioxidative status in piglets among L-Gln treatments may be related to the differences like feed intake, blood urea nitrogen and other factors that ultimately result from Gln treatments since the organism is complicated. Together, L-Gln improved the antioxidative capacity in piglets by an increase in the synthesis of GSH.

Gut microflora play a critical role in regulating host health, and the disruption of gastrointestinal microbial structure may result in a series of metabolic disorders or even diseases (Thaiss et al., 2016). Intestinal microbiota can be affected by multiple factors like the use of antibiotics, diseases, weaning and dietary changes, during which the structure of the gut microbiota may alter, making pigs vulnerable and predisposed to infections (Summers et al., 2019). In the present study, weaned piglets fed low-protein diets supplemented with L-Gln, a conditionally essential AA, had similar results regarding alpha diversity among the treatments, indicating that L-Gln administration exerted little effect on microbial composition diversity. We also found that piglets fed a diet with 1% L-Gln had higher abundance of p_75_a5, Gemmiger, Lactobacillus, Clostridium, and Prevotellaceae_Prevotella, with the abundance of Streptococcus being concomitantly downregulated on d 14. Likewise, dietary 1% L-Gln upregulated the genus abundance of p_75_a5 and Gemmiger in the stool of piglets on d 28, indicating 1% L-Gln administration optimized the microbial structure as Gemmiger and Lactobacillus have been widely considered probiotics (Zhou et al., 2023), and Prevotellaceae has been shown to ameliorate PD-1/PD-L1 inhibitor-related cardiotoxicity in colonic macrophages by producing butyrate (Chen et al., 2022). The inconsistencies in microbe alterations at genus level among the L-Gln treatments may be related to feed intake, both directly and indirectly. In agreement with our study, Xu et al. (2021) demonstrated that Gln ameliorated DSS-induced reduction of Lactobacillus in the colonic content of mice. Consistently, pregnant sows fed a diet supplemented with 1.0% Gln had higher abundance of Bacteroidales, and Prevotella (Zhang et al., 2017b), suggesting that Gln has a regulatory effect on microbial metabolism in the gastrointestinal tract. KEGG pathways further revealed that L-Gln administration could decrease cell apoptosis, promote DNA replication, enhance the metabolism of Gln, Glu, and pyrimidine, and prevent various human diseases like cancer, cardiovascular disease, endocrine and metabolic disease, infectious disease, and neurodegenerative disease on d 14. Consistently, Gln could promote DNA synthesis of intestinal epithelial cells by activating the ornithine decarboxylase (Wu, 1998). However, the functions of hypertrophic cardiomyopathy, platinum drug resistance, pathogenic E. coli and Shigellosis infection were promoted in response to 2% and 3% L-Gln administration on d 28. Among them, E. coli and Shigellosis infection are critical factors in intestinal disease etiology, indicating a high level of L-Gln could bring detrimental effects to piglets. Still, the role of gut microflora mediated by L-Gln is lacking in the context of low-protein diets. Therefore, the regulatory mechanism of L-Gln on gut microbiota in piglets fed with low-protein diets needs to be investigated in the future.

In summary, we found that supplementing 1% L-Gln improved the growth performance, serum physiochemical parameters, and antioxidative capacity in the low-protein diet-fed piglet at an early age. These beneficial effects of L-Gln were associated with an increased synthesis of GSH by modulating AA levels, and the optimization of intestinal microbial structure (like upregulating the abundance of Gemmiger and Lactobacillus and reducing the relative level of Streptococcus) instead of microbial composition diversity. Moreover, 1% L-Gln prefers to exert its functional effects on antioxidation and modulation of intestinal microbiota rather than be a single fuel in low-protein diet-fed pigs at an early age. Of note, a low-protein diet supplemented with 2% or 3% L-Gln compromised the growth performance in weaned piglets, which is related to the accumulation of blood urea nitrogen, inhibitory metabolism of arginine and proline, and increased susceptibility to E. coli and Shigellosis infection.