INTRODUCTION
In recent years, environmental regulations on the swine industry have become increasingly stringent due to the intensifying impacts of climate change and environmental pollution [1,2]. Among the various factors contributing to the environmental footprint of pig production, nitrogen excretion has received considerable attention because of its adverse effects, including water pollution, acidification, and greenhouse gas emissions [3]. Moreover, excessive dietary nitrogen intake through high crude protein (CP) levels not only reduces feed efficiency but also disrupts intestinal microbiota balance, increases odor emissions, and elevates the risk of diarrhea [4]. To address these issues, numerous studies have focused on reducing dietary CP levels by supplementing essential amino acid (AA), and maintaining nutrient balance while minimizing nitrogen excretion [5,6]. In particular, arginine supplementation has gained attention for its potential to optimize AA profiles, support intestinal development, and enhance immune function [7,8]. However, the practical effects of arginine supplementation on growth performance remain limited. This is primarily due to its short half-life, competitive interactions with other AA such as lysine, tryptophan, and histidine, and its susceptibility to degradation by arginase. Additionally, arginine is highly degraded by intestinal microbial metabolism [9], which may further compromise its bioavailability. Therefore, further research is warranted to overcome the limitations of arginine supplementation and to develop strategies that effectively incorporate arginine into low-CP diets without compromising pig health or performance.
N-carbamylglutamate (NCG) has recently attracted considerable attention as a promising feed additive due to its role as a precursor of arginine [10,11]. Structurally, NCG is analogous to N-acetylglutamate, which serves as a critical cofactor for carbamoyl phosphate synthetase I as an essential enzyme in the urea cycle [12]. In addition to its functional similarity, NCG exhibits favorable physicochemical properties, including high mitochondrial permeability and a longer half-life compared to arginine, making it suitable for nutritional applications [9,13]. These characteristics enable NCG to enhance endogenous arginine synthesis, which in turn promotes the production of important antioxidant metabolites such as nitric oxide (NO) and polyamines [14]. The greater systemic antioxidant capacity may support the growth performance of the recent fast-growing pigs in high stocking density in commercial farms. Therefore, it was reported that dietary supplementation with NCG has been associated with reduced ammonia emissions and improved nitrogen utilization [15,16]. In weanling pigs, NCG has been shown to alleviate weaning stress by enhancing growth performance and supporting intestinal development and health [17,18]. Furthermore, in growing-finishing pigs, NCG supplementation has been linked to improvements in carcass quality and meat characteristics.
Despite these promising outcomes, the effects of NCG in early growing pigs remain relatively underexplored. Therefore, the present study was conducted to evaluate the efficacy of NCG supplementation on growth performance, nutrient digestibility, blood AA profiles, and intestinal enzyme activities in early growing pigs.
MATERIALS AND METHODS
The experiment was conducted on a commercial pig farm in Haman, Korea. Growing pigs with an average initial body weight (BW) of 20.38 ± 2.65 kg of the Landrace × Yorkshire × Duroc breeds were housed in partially slatted concrete floor pens equipped with feeders and nipple drinkers. Feed and water were given as ad-libitum. Pigs were placed in a pen of 2.80 × 5.00 m diameter. The experimental environment was controlled at 24°C of temperature and 70 % of relative humidity by an automatic environment control system. A total of 240 crossbred grower pigs were allotted based on their initial BW in four treatments. Treatments were divided by dietary CP level (16% of moderate CP [MCP] in diet; 14% of low CP [LCP] in diet) and NCG supplementation (without NCG [WN]; 0.1% NCG supplementation [NS]). NCG (≥ 97% purity) was sourced from Beijing Animore Sci & Tech. Each treatment had 10 replicates, with six pigs (3 males and 3 females) per replicate, over a 4-week period. The experimental diet was formulated to meet the nutritional requirements of pigs recommended by NRC [19] (Table 1).
Supplied per kg of diet: 16,000 IU vitamin A (palmitate), 2.00 mg vitaminB1 (thiamin), 5.00 mg vitamin B2 (riboflavin), 2.00 mg vitamin B6 (pyridoxine), 0.03 mg vitamin B12 (cyanocobalamin), 25.00 mg niacin, 0.40 mg folic acid, 0.05 mg biotin, 5.00 mg ethoxyquin, 2,000 IU vitamin D3 (cholecalciferol), 75.00 mg vitamin E (dl-α-tocopheryl acetate), 2.00 mg vitamin K3 (menadione).
The experiment assessed growth performance at the start and end of each phase by examining data related to average daily gain (ADG), average daily feed intake (ADFI), BW, and feed efficiency (G:F). The G:F was determined through the computation of the ADG divided by the ADFI. Throughout the entire experiment, mortality was monitored, but no instances were recorded.
Digestibility of dry matter (DM), CP, gross energy (GE), and ether extract (EE) was determined using chromium oxide (Cr2O3) as markers. For 7 days prior to the end of the experiment, growing pigs were fed powder feed mixed with 2.5 g/kg Cr2O3. Fecal samples were collected on day 3 before the end of the trial and mixed in each pen to obtain a homogeneous representative sample; then a subsample was taken and frozen at –20°C for further examination. Feed and fecal samples were analyzed for nutrient digestibility. Fecal samples were thawed and dried for 72 h in an Advantec FC-610 oven (Toyo Seisakusho) operating at 75°C for the evaluation of DM content. Before proximate analysis, the dried fecal samples were first ground to a suitable size through a 1-mm filter (Christy and Norris Hammer Mill). Estimation of CP content was made using method 990.03 [20]. A Soxhlet extraction (Soxtec 2050, FOSS North America) was used for EE analysis using method 954.02 [20]. A bomb calorimeter (Parr 1261 bomb calorimeter, Parr Instruments) was used to evaluate GE content.
At the end of this experiment, pigs were euthanized to take samples for apparent ileal digestibility (AID) of AA. The digesta samples were taken from the distal part of the ileum and stored in the refrigerator at –20°C until the analysis. The digesta samples underwent a thawing process prior to freeze-drying. The determination of AA was done by hydrolyzing samples in glass tubes sealed under vacuum for 24 h with 6 N HCl (including phenol) at 110 ± 2°C. Methionine and cysteine analysis were determined after oxidation with performic acid. The identification of AA was conducted using a Waters ion-exchange High-Performance Liquid Chromatography system, and the software (version 3.05.01, Millennium, Waters, and Millipore) was used to integrate the chromatograms following the method established by Hosseindoust et al. [21].
In the equations provided,
Ndiet and Ndigesta represents the nutrient concentrations of the diet and digesta, respectively (% of DM); Crdiet and Crdigesta are the Cr2O3 of the diet and output, respectively (% of DM).
Digesta enzyme activity of trypsin, chymotrypsin, protease, amylase, and lipase activity was analyzed from all pigs following the assay kit instructions. Trypsin (Abcam Limited, catalog No. ab102531), Chymotrypsin (Abcam Limited, catalog No. ab234051), Protease (Abcam Limited, catalog No. ab111750), Amylase (Abcam Limited, catalog No. ab102523), and Lipase (Abcam Limited, catalog No. ab102524).
Blood samples (10 mL each) were collected from two selected pigs per pen from the middle range of BW in the group. Sampling was performed via vena cava venipuncture using disposable Vacutainer SST II Plus plastic serum tube (367955, Becton Dickinson). The serum was then separated from the blood samples by centrifuging them for 15 minutes at 4°C at 3,000×g. After that, the blood sample was stored at –20°C until analysis. Blood urea nitrogen (BUN) levels were determined using a Cobas 6000 analyzer (Roche Diagnostics). The analysis was performed using the kinetic/potentiometric method, which measures the rate of change in absorbance at a specific wavelength to quantify the concentration of BUN in the serum samples.
Data generated in the present study were statistically analyzed by adopting two-way ANOVA using the GLM procedure by the SAS (SAS Institute). Factorial arrangement was analyzed by dietary CP level and NCG supplementation. Significant differences between treatment means were identified using Tukey’s honest significant difference test. Data were expressed as means and standard deviations, with values of p < 0.05 classified as statistically significant, and values of 0.05 ≤ p < 0.1 were considered as a tendency.
RESULTS
Pigs fed the MCP diet tended to have a higher final BW compared to those on the LCP diet (p = 0.088), although the difference was not statistically significant. NCG supplementation did not significantly affect final BW (Table 2). The ADG was greater in the MCP group than in the LCP group (p < 0.05). Similarly, the G:F was significantly improved in pigs fed the MCP diet (p < 0.05), and showed a trend towards improvement with NCG supplementation (p = 0.089). The ADFI was not influenced by either dietary CP level or NCG supplementation. No significant interaction between dietary CP level and NCG supplementation was observed for any growth performance parameter.
The CP digestibility tended to be higher in pigs receiving NCG supplementation (NS) compared to those without NCG (WN) (p = 0.093; Table 3). However, the digestibility of DM, GE, and EE was unaffected by NCG supplementation. The dietary CP level had no significant effect on nutrient digestibility.
The AID of arginine and tyrosine was significantly greater in the NS group than in the WN group (p < 0.05; Table 4). Additionally, AID of lysine (p = 0.067), glycine (p = 0.058), and the average of essential AA (p = 0.091) showed trends towards improvement with NCG supplementation. The AID of serine and the average of non-essential AA exhibited significant interaction effects between dietary CP level and NCG supplementation (p < 0.05).
Trypsin activity in the jejunal digesta tended to increase in the NS group compared to the WN group (p = 0.087; Table 5). However, the activities of chymotrypsin, protease, amylase, and lipase were not significantly affected by dietary CP level or NCG supplementation.
The LCP diet significantly reduced lysine and alanine concentrations in the blood compared to the MCP group (p < 0.05; Table 6). Furthermore, BUN (p = 0.073), arginine (p = 0.069), valine (p = 0.053), and the average essential AA concentration (p = 0.063) tended to be lower in the LCP group. The NS group exhibited significantly higher concentrations of arginine, lysine, essential AA, non-essential AA, and total AA in the blood (p < 0.05). Significant interaction effects between dietary CP level and NCG supplementation were observed for arginine and essential AA concentrations (p < 0.05).
DISCUSSION
The results of the present study demonstrated a beneficial trend of NS in enhancing feed efficiency when incorporated into the LCP diet for early growing pigs. The early growing period is a critical phase that demands rapid muscle accretion to ensure optimal transition into the finishing stage [6,22]. However, LCP diets, although effective in reducing nitrogen excretion and odor emissions, may fail to supply sufficient protein and AA to support ideal growth and muscle development [23]. NCG, a functional analog and metabolic precursor of arginine, plays a pivotal role in promoting muscle protein synthesis [10]. NCG also serves as a precursor for mammalian target of rapamycin (mTOR), a protein kinase that stimulates muscle protein synthesis and reduces BUN levels [24,25]. Additionally, NCG-derived NO has been shown to enhance glucose uptake and lipolysis and support energy metabolism and overall growth [26]. In line with our findings, Li et al. [24] reported that supplementation with 0.08% NCG in finishing pigs improved ADG even when dietary CP was reduced by 2.4%. Similarly, Hu et al. [18] observed that 0.05% NCG supplementation in weaning pigs enhanced growth performance during the experimental period. Collectively, these results suggest that NCG supplementation in LCP diets may enhance crude protein utilization and support muscle synthesis, ultimately contributing to improved feed efficiency in early growing pigs.
In the present study, no significant differences were observed in the apparent digestibility of DM, GE, and EE among dietary treatments. However, CP digestibility tended to be higher in the NS group compared to the WN group. Nutrient digestibility in pigs is influenced by multiple physiological factors, including the composition of the intestinal microbiota, gut morphology, expression of tight junction proteins, and digestive enzyme activity [27,28]. NCG has been reported to enhance intestinal development by promoting the epithelial cell function and maintaining structural integrity [16]. Furthermore, it has been shown to increase villus height and upregulate the expression of tight junction proteins, thereby supporting efficient nutrient absorption [18]. These effects are likely associated with NCG’s role to enhance antioxidant capacity via NO production, particularly by stimulating the development and function of intestinal mitochondria [29]. Our study also suggests that NCG supplementation improves nutrient digestibility through the enhancement of intestinal health and development. The AID of arginine and lysine was greater in the NS group compared to the WN group. Both arginine and lysine are primarily absorbed in the jejunum via AA transporters, particularly members of the solute-carrier family [15]. NCG supplementation is known to enhance the expression of these AA transporters by stimulating the production of goblet cells and mucin secretion, thereby strengthening the intestinal mucosal barrier [18,30]. Wang et al. [16] demonstrated that the inclusion of NCG in a diet with 6% reduced crude protein significantly improved the AID and bioavailability of AA in growing pigs. In our study, jejunal trypsin activity was elevated in the NS group. Trypsin, a key proteolytic enzyme involved in the digestion of arginine and lysine, is activated by enterokinase, an enzyme secreted by the duodenal mucosa [31]. The observed increase in trypsin activity may be attributed to the stimulatory effect of NCG on intestinal mucosal proliferation, possibly mediated through activation of the mTOR pathway and subsequent protein synthesis within intestinal epithelial cells [32]. NCG supplementation may indirectly supports intestinal barrier function while activating the antioxidant status in gut mitochondria.
In this study, BUN levels decreased as dietary CP levels were reduced. However, NCG supplementation did not exert an additional effect on this reduction. BUN is a metabolic by-product of protein degradation and serves as a reliable indicator of protein metabolism and nitrogen utilization efficiency in pigs [33]. NCG has been reported to reduce BUN levels by enhancing nitrogen metabolism and facilitating more efficient excretion of nitrogenous waste. Wang et al. [34] observed that BUN concentrations were significantly reduced with NCG supplementation when pigs were fed diets with identical CP levels. In contrast, Li et al. [27] reported that NCG supplementation in low-CP diets did not affect BUN concentrations compared to low-CP diets without NCG. These findings suggest that the effect of NCG on BUN concentration may be influenced by the overall dietary protein and AA content. In addition to its potential effect on nitrogen excretion, NCG supplementation in the current study was associated with increased serum concentrations of arginine, lysine, and isoleucine. The measurement of serum AA levels may provide a more sensitive indicator of the metabolic efficacy of NCG than BUN alone. Compared to arginine, NCG possesses a longer half-life, which may contribute to sustained stimulation of mitochondrial function and activation of the mTOR signaling pathway, which are critical regulators of protein synthesis [9,13]. Consistent with our findings, Wang et al. [34] also reported that NCG supplementation increased circulating AA levels in growing pigs, further supporting the notion that NCG can enhance systemic AA availability and utilization. Taken together, these results suggest that NCG may play a beneficial role in optimizing protein metabolism and AA efficiency, particularly under conditions of reduced dietary CP.
CONCLUSION
The findings of this study suggest that NS supplementation in LCP diets tended to improve feed efficiency in early growing pigs. NCG also tended to enhance crude protein digestibility and significantly increased the apparent ileal digestibility of arginine and lysine. These effects may be attributed to the stimulatory role of NCG on circulating amino acid concentrations. Overall, the results highlight the potential of NCG to improve growth-related physiological parameters, particularly under protein-restricted dietary conditions.
