With the shortage of feed resources and further aggravation of environmental pollution, it is difficult to increase animals' food production by simply expanding the number of breeding heads and ignoring environmental pollution. Technological innovation is one of the main ways to transform animals' production mode and improve animals' output rate and resource utilization rate, which are beneficial for establishment of resource-saving and environment-friendly society in the future (1). A large amount of nitrogen substances excreted by livestock is an important cause of environmental pollution in animal husbandry (2, 3). These nitrogen substances mainly derive from the undigested crude protein and degradation of amino acids in feed (4, 5). Nitrogen is one of the essential components of proteins and nucleic acids and plays a crucial role in the growth and development of ruminants. However, under the current dietary feeding system, the nitrogen utilization rate of ruminants is lower than that of monogastric animals (6). The nitrogen utilization rate of ruminants is approximately 20 to 36%, and the remaining 64 to 80% of nitrogen is excreted in the form of feces and urine (7), which not only reduces protein conversion and utilization, but also increases manure nitrogen discharge (8–10). Lower nitrogen utilization of ruminants severely restricts the economic benefits of livestock farms. Therefore, increasing the efficiency of nitrogen conversion and utilization in ruminants by nutritional strategy is of great significance for ruminants industry as well as reducing the environmental pollution (11).

Hippuric acid (HA), also known as benzoylglycine, is the glycine conjugate of benzoic acid (BA), and it is found in high concentrations in the urine of herbivores (12). For herbivores, including ruminants, polyphenols in plant-based diets can form BA under the joint action of gut microorganisms, which are subsequently absorbed through the gut and transported to the liver for metabolism (13). In animals' liver mitochondria, BA and glycine are catalyzed by enzymes to form HA, which is then excreted in the urine (14). In early research, Doak (15) found that urea nitrogen accounted for 76.4% of urinary nitrogen in wether, and the proportions of allantoin nitrogen and HA nitrogen were 4.1 and 2.6%, respectively. Bristow et al. study analyzed the nitrogen content in the urine of cattle and sheep, and found that 69% of the total nitrogen in bovine urine was in the form of urea, 7.3% in the form of allantoin, and 5.8% in the form of HA. In sheep urine, the nitrogen in the form of urea accounted for 83% of the total nitrogen content, and both HA nitrogen and allantoin nitrogen accounted for 4.3% of total nitrogen (16). Thus, nitrogen excreted by HA can be used as another way of nitrogen excretion in ruminants.

BA is an important precursor in the process of HA formation, and its content directly affects the excretion of HA. As a weakly acidic aromatic acid organic compound, BA is widely used as organic acidifier and preservative in food, medicine and feed industry because of its broad antibacterial effect and strong antibacterial ability (17, 18). BA can improve the growth performance and feed conversion of monogastric animals by promoting the production and activation of gastrointestinal digestive enzymes (19), enhancing gut absorption capacity (20), improving intestinal barrier (21) and regulating intestinal microbiota (22). Nevertheless, little attention has been paid to the effects of BA on urea metabolism, nitrogen metabolism and growth performance of ruminants. Noticeably, both HA synthesis and urea cycle occur in the liver mitochondria of ruminants. Among them, the urea cycle mainly consists of 1 molecule NH₃ and 1 molecule CO₂ catalyzed by carbamyl phosphate synthetase I to produce carbamyl phosphate (23). The NH 4+ produced by glutamine in mitochondria can be used to synthesize carbamyl phosphate as well as glycine. When the entry of BA into liver cells increases, HA synthesis is elevated, and the consumption of glycine correspondingly increases, thus up-regulating the synthesis of glycine in liver mitochondria. If the amount of NH 4+ in the mitochondria used to synthesize glycine increases, does the amount used to synthesize carbamyl phosphate decrease, then reducing urea production? However, there is a lack of relevant research at present. Therefore, this study was carried out to evaluate the effects of different levels of BA supplementation on nutrient digestibility, urea metabolism, nitrogen metabolism and growth performance of weaned lambs.

Lamb nutrition is an increasingly important issue in today's sheep production. The feeding management of lambs has long-term influence on future production performance of meat quality of fattening sheep (31). Due to the immature gastrointestinal tracts, the lambs are easily affected by harmful microorganisms. After weaning, lambs undergo the changes of feed type and rearing pattern, resulting in decreased nutrient digestibility and growth rate (32). Relieving the weaning stress of lambs is of great significance for improving the healthy growth of lambs. As an aromatic carboxylic acid, BA has multiple health benefits, including antibacterial and anti-inflammatory activities (18). Previously, a research has found that dietary supplementation of BA could increase ADG of weanling pig, but had no obvious difference of feed efficiency (33). In the current study, 1% BA supplementation significantly increased ADG and feed efficiency when compared to 0 BA and 1.5 BA groups. The different reason may be that the animals used in experiment was different. BA can produce esterification products with bacteria, which then affect the metabolism of pathogenic bacteria. Besides, BA can interfere with the DNA activity of pathogenic bacteria, thereby decreasing their growth (34). These effects are conducive to reducing the incidence rate of diarrhea and relieving the negative impact on growth rate of lambs caused by weaning stress. Our results showed that appropriate addition (1%) of BA improved growth performance of weaned lambs. The improved growth performance of animals is usually associated with higher apparent digestibility and nitrogen metabolism. Hence, we conducted the following experiment to study the influence of BA on apparent digestibility and nitrogen metabolism of weaned lambs.

The gastrointestinal tracts of weaned lambs have insufficient digestive enzyme secretion, thus the nutrients digestibility of lambs are low (35). In our study, 0.5 and 1% dietary supplementation of BA significantly increased the nutrients intake of lambs. In general, the typical feeds fed with young ruminants have relatively high values of acid binding capacity, which affect the nutrients intake of animals (36). A previous study found that dietary supplementation with BA could decrease acid binding capacity of feeds (37), which might explain the positive effects of BA on nutrients intake. The digestibility of DM and OM are key parameters to reflect the utilization ability of feed by animals (38). In the current study, the DM and OM digestibility in 1 BA group showed highest values and were significantly elevated as compared with 0 BA group, indicating that the lambs given to 1% BA could obtain more nutrients and accelerate growth, which were matched to ADG results. Organic acid is an effective alternative to enhance nutrient digestibility in animals industry production. The organic acid has multifunctional effects, including reduction of gastrointestinal pH, enhancement of gastrointestinal retention time, stimulation of pancreatic secretions and promotion of gastrointestinal morphology, thus improving nutrient digestibility (39, 40). Our results might relate to the ability of BA to regulate the gastrointestinal pH and digestive enzyme levels and improve gut morphology. In the future, the effects of BA on gastrointestinal development of lambs need in-depth investigation. In addition, we also found that the NDF and ADF digestibility of 1 BA group were higher than 0 and 1.5 BA groups. In ruminants, the microbial community in the rumen is responsible for crude fiber utilization. Unfortunately, the effect of BA on ruminal microbial community has not been reported. A recent study verified that dietary supplementation of BA could stabilize the microbiota fluctuation caused by weaning stress and speed up rapid colonization of dominant bacteria in the gut of piglets (41). Even though the microbial community between monogastric animals and ruminants existed difference, these results indicated that BA could affect the microbial community. We speculated that the positive effects of BA on NDF and ADF digestibility might be associated with the regulation of microbiota in the gastrointestinal tracts. Lastly, the activity of digestive enzyme plays an important role in the nutrients digestion (42). Therefore, more experiments are needed to explore the effects of BA on microbial community and digestive enzyme activity of weaned lambs.

Previously, studies have reported that with the elevation of dietary BA supplementation, the urinary pH was linearly reduced (43), and besides, the HA concentration in urine was linearly increased (44, 45), which also displayed similar findings in our research. The reason for reduced urinary pH may be due to the elevation of urinary HA content. After absorption, the BA is conjugated with glycine through glycine-N-acylase, and then transformed into HA in the liver (46). Moreover, the urea nitrogen concentration in 1% BA supplementation treatments was significantly decreased (3 h), suggesting that the nitrogen conversion of 0 BA lambs was low. As mentioned earlier, the HA synthesis and urea cycle occur in the hepatic mitochondria of ruminants, and together consume NH 4+ produced from dietary amino acid metabolism (47). Thus, when BA enters the animal's body, does the increase of HA synthesis have the effect of reducing urea production? In the current study, results showed that with the increase of BA intake, the urinary HA content and HA excretion increased linearly, while urinary urea nitrogen content decreased linearly. At the same time, it was found that there was a significant correlation between urinary urea nitrogen excretion and HA nitrogen excretion. This result confirmed our previous hypothesis that the elevation HA excretion by adding BA to the diet reduced urea nitrogen excretion. However, there were unsatisfactory results in terms of total urinary nitrogen output. In addition to hippuric acid nitrogen and urea nitrogen, there are also nitrogen produced by purine derivatives in urinary total nitrogen. In previous studies of crossbred Bulls, it was found that nitrogen produced by purine derivatives (such as allantoin nitrogen) increased linearly as feed intake increased (48). In our experiment, other component nitrogen excretion of weaned lambs increased significantly, and the unsatisfactory total urinary nitrogen excretion might be due to the higher feed intake of 0.5 and 1% BA lambs. In addition, according to our unpublished data, after feeding BA to lactating lambs, metabolites of jejunum contents were mainly enriched in pyrimidine and purine metabolism pathways, indicating that BA intake also affected the metabolism of purine derivatives in lambs. A previous study in rabbits found that intake of sodium benzoate led to an increase in urinary HA excretion and a decrease in urinary urea nitrogen excretion (49), which were consistent with the results of our experiment. Nousiainen et al. (50) reported that increased content of urinary and blood urea nitrogen, indicated a reduced nitrogen efficiency of cows. The 0.5 and 1 BA groups showed higher nitrogen intake when compared to 0 and 1.5 BA groups. According to our results, appropriate supplementation of BA in the diet could improve nitrogen utilization of weaned lambs, which was helpful for promoting growth.

Recently, a study reported that the improvement of ADG was attributed to the elevated nitrogen retention in pigs fed diets with BA (51). In the current study, we did not find obvious difference of nitrogen retention among all groups. However, the 1 BA group had highest value of nitrogen retention. A previous study found that dietary supplementation of BA could improve the intestinal morphology and up-regulate the expression of glucagon-like peptide 2 gene in the intestinal mucous (37), which might enhance nitrogen retention in animals fed ration with BA. We also found that the addition of 1.5% BA to the basal ration decreased nitrogen intake and reduced nitrogen utilization in lambs, thus negatively affecting growth performance. The possible reason might be related to elevation of glycine synthesis required to transform BA into HA in the liver, which might affect the amino acid metabolism and reduce amino acid concentration for synthesis of protein (46). Excessive utilization of protein and amino acid for transformation of HA reduces nitrogen retention. Thus, according to our results, the appropriate supplementation level of BA in the diet of lambs was 1%.

As important indexes related to health condition of animals, the changes of serum biochemistry can be utilized to estimate the body's physiological metabolism and organ functions (52). The T-Bil, D-Bil, ALT, AST, ALP and GT contents in plasma can be used to evaluate the liver function, and the changes of GLU, TG and TC concentrations are associated with fat metabolism. Furthermore, the plasma concentrations of TP and ALB are key parameters of protein metabolism (52). In the present experiment, the plasma contents of GLU, T-Bil, D-Bil, ALT, AST, ALP, GT, TP, TG and TC were similar among four treatments, suggesting that BA supplementation did not have adverse effects on lipid metabolism and hepatic function of weaned lambs. A previous study found that BA supplementation significantly increased the contents of ALB in blood, and reduced the urea nitrogen content (53). Consistent with previous study, our study found that 1% BA supplementation increased plasma ALB content and reduced urea nitrogen concentration, indicating that BA could improve the anabolism of protein to a certain degree, which was conducive to promoting growth of lambs.

Previous studies have verified that the HA is formed by BA biosynthesis reaction and can be excreted from the body's urine within 4 h (54, 55). We studied the dynamic changes of BA, HA and urea nitrogen in plasma of weaned lambs fed BA for the first time. Results showed that after feeding BA 1 h, the plasma concentrations of BA and HA reached up to highest value and then decreased gradually, and the 1 and 1.5 BA groups were higher than other groups. BA is absorbed through the animal's intestine and enters the bloodstream, where it is then transported to the liver for metabolism (13). Our findings indicated that after BA transported to the liver, the process of BA and glycine producing HA under the catalysis of enzyme was very rapid. In animals, BA and glycine are catalyzed by enzymes to form HA, which is then excreted in the urine. In addition, BA can also be metabolized by gut microbes (56, 57). The microbial community of lambs were still in developmental stage, and higher levels of BA and HA may have positive effects on microbial community. However, the potential mechanism of action still needs exploration. As the main products of protein metabolism of animals' body, urea nitrogen are important indexes to reflect protein utilization. The decrease of these contents indicates that the protein utilization is improved, which contributes to nitrogen deposition, and besides, high urea nitrogen content can also have adverse effects on animal health (58). In this study, dietary supplementation of BA effectively decreased plasma urea nitrogen content after morning feed from 2 to 3 h, which was in line with urinary urea nitrogen content mentioned earlier. This result was conducive to improving nitrogen metabolism of lambs. Future research should be paid more attention to the potential mechanisms of BA on the nitrogen metabolism of lambs.

Taken together, BA supplementation had the ability to improve nitrogen metabolism of waned lambs, which was conducive to promoting growth of lambs. However, some limitations need to be acknowledged in the current study. First of all, the absorption pathway of BA in the gastrointestinal tracts requires further elucidation. In the subsequent experiment, we will explore the potential mechanism of action using in vitro and in vivo studies. In addition, future studies to understand the functions of microbial community in the gastrointestinal tracts using metagenomics or culturomics as well as host function using transcriptomics are needed to provide more information on the role of microorganism in the digestive organ of weaned lambs and their response to dietary BA supplementation.

Supplementation of BA (1%) improved the ADG, feed efficiency and DM, OM, NDF and ADF digestibility of weaned lambs. In addition, dietary supplementation with 1% BA significantly increased urinary HA contents and excretion as well as nitrogen intake. After supplementation with BA, the plasma and urinary urea nitrogen contents were reduced. Thus, based on our findings, the appropriate supplementation of BA (1%) in the diet improves the growth performance and nitrogen metabolism of weaned lambs.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

All procedures involving animal care and management used in this experiment were authorized (protocol number: 2020022) by the Institutional Animal Care and Use Committee of Xinjiang Agricultural University (Urumqi, Xinjiang, China). The study was conducted in accordance with the local legislation and institutional requirements.

WZ: Conceptualization, Investigation, Visualization, Writing – original draft, Writing – review & editing. SS: Investigation, Visualization, Writing – review & editing. YaqZ: Investigation, Writing – review & editing. YanZ: Writing – original draft. JW: Investigation, Writing – review & editing. ZL: Investigation, Writing – review & editing. KY: Conceptualization, Data curation, Funding acquisition, Methodology, Project administration, Supervision, Writing – review & editing.