Introduction

Beyond their fundamental role in protein synthesis, arginine exhibits many important biological functions within organisms (Narita et al. 1995). Arginine is the only nitrogen precursor for producing nitric oxide (NO), a key endogenous signaling molecule (Rahimnejad and Lee 2014). Arginine is also a precursor for polyamine synthesis, which plays a diverse range of biological functions in vivo, including the regulation of protein synthesis, cell growth and differentiation, gene expression, neurotransmission, and immune regulation (Zheng et al. 2019; Han et al. 2018). Moreover, arginine plays a crucial role in regulating endocrine and reproductive functions, as well as extracellular signal pathways including AMP-activated protein kinase (AMPK) and rapamycin targets (TOR) (Chen et al. 2012; Fuentes et al. 2013; Seiliez et al. 2008).

The synthesis of arginine in mammals has been extensively studied and documented (Wu and Morris 1998). In mammals, intestinal cells can convert glutamine and glutamic acid into ornithine, which is then converted into citrulline. The kidneys can further convert citrulline molecules into arginine (Flynn and Wu 1996; O’Sullivan et al. 1998; Wu et al. 1997). However, under certain physiologic and pathophysiologic conditions, endogenous synthesis of arginine is insufficient to meet the body’s requirement. In such situations, dietary supplementation of arginine becomes necessary. For example, studies focusing on piglets have demonstrated that endogenous synthesis of arginine can only meet 50-69% of their daily arginine needs (Wu et al. 1997; Wu and Knabe 1995).

Fish, especially carnivorous species, heavily rely on amino acids for ATP production, which results in significant amino acid catabolism and low retention rates compared to mammals (Li et al. 2020b). Previous studies have shown that endogenous synthesis of arginine is limited in teleost fish (Andersen et al. 2016; Korte et al. 1997; Wright 2011). The low or no activities of P5CS (pyrroline-5-carboxylate synthetase), OAT (ornithine aminotransferase), and CPS III (carbamoyl phosphate synthetase III) have observed in teleost fish, with ammonia being the primary form of nitrogen excretion (Wood et al. 1989). Therefore, the hypothesis proposing the elimination of the urea cycle during the evolution of teleost fish is widely accepted (Wood et al. 1989). However, previous research has shown that oral administration of gabaculine (an OAT inhibitor) significantly reduces the concentrations of citrulline and arginine in the bloodstream of channel catfish (Ictalurus punctatus) juveniles (Buentello and Gatlin 2001). These findings indicated that endogenous biosynthesis of arginine plays a crucial role in maintaining the dynamic balance of arginine in juvenile channel catfish. However, in the absence of relevant research, the specific pathway of arginine metabolism in fish has not yet been elucidated.

Largemouth bass (M. salmoides) is a highly valuable fish in farming due to its delicious flesh and fast growth (Guo et al. 2019). As a representative carnivorous species, largemouth bass is important species for studying amino acid metabolism in fish. Previous research has shown that largemouth bass possess all the genes required to synthesize urea cycle enzymes (Kong et al. 1998). However, there are low levels of serum arginine in largemouth bass (Li et al. 2020a). One of our previous study investigated the effects of dietary arginine family amino acids supplementation on growth, whole-body amino acid profiles, antioxidant capacity, and gene expression of juvenile largemouth bass (Luan et al. 2025). The specific function and mechanism of the urea cycle in largemouth bass are not fully understood. To address this knowledge gap, this study focuses on investigating the metabolism of arginine in largemouth bass juveniles after oral or intraperitoneal administration of arginine or its substrates, and aiming to provide new insights into the metabolism of arginine in fish.

Materials and methods

Animal management

Largemouth bass fry was bought from Zhejiang Zhengda Aquaculture Company and raised for 12 weeks in ten 2000 L polyethylene tanks at the Nutrition and Feed Laboratory of Zhejiang Ocean University. Fish are fed a homemade feed twice daily (08:00 and 17:00). The feed contained 46% protein (Anderson et al. 1981) and 12% lipid (Bright et al. 2005) to meet the nutritional requirements. Each tank was supplied with municipal water that has been dechlorinated for 24 h, and provided aeration. During the rearing period, a 12-hour light / 12-hour dark photoperiod was maintained, and the water temperature was kept between 24–26℃. Water quality parameters were maintained within the appropriate range, including ammonia nitrogen (NH3 and NH4+) concentration below 0.05 mg / L, pH value of 7.00 ± 0.2, and nitrite (NO2−) concentration below 0.01 mg / L.

Once fish reached the desired experimental size (average weight of 25 ± 0.5 g), the rearing experiment was concluded. After fasting 24 h, the fish were anesthetized using MS-222 (ethyl 3-aminobenzoate methane sulfonate, Aladdin, Shanghai, China) at a concentration of 150 mg / L for 5 min. After being weighed individually, 300 healthy fish were selected and transferred to new polyethylene tanks where they would not receive any food throughout the duration of the experiment.

Experiment reagent

The amino acid concentration of all injected solutions was 0.66 mol / L. L-arginine hydrochloride, arginine-aspartate, and L- citrulline were purchased from Aladdin (Shanghai, China) and dissolved directly in 0.9% sodium chloride solution. The pH of the solution was adjusted to 7.5 using hydrochloric acid. L-glutamate (Aladdin, Shanghai, China) was dissolved in 0.9% sodium chloride solution containing hydrochloric acid, and then the pH was adjusted to 7.5 using sodium hydroxide solution. The configured injection solution was autoclaved (121 °C) for 30 min. The purity of all amino acids used in the test was 98%.

Oral administration test

In this experiment, 150 fish were used and divided into five groups, with each group containing 30 fish. These fish were given different solutions through oral administration, which included 0.9% sodium chloride, L-arginine, arginine-aspartate, citrulline, and L-glutamate. They were anesthetized with MS-222 for 5 min at a concentration of 150 mg / L before the oral administration. The dosage of the oral amino acid solution was based on the weight of the fish (10 µL / g). The solutions were injected directly into the fish’s stomachs using sterile disposable syringes. After the oral administration, each fish was placed in a 20 L polyethylene bucket filled with dechlorinated water and provided with aeration for better survival. Each group was divided into five time points after oral administration for sampling (0, 10, 30, 60, 120, 240 min, respectively).

At each time point, 6 fish were randomly selected from each group for sample collection. The samples from the 6 fish were combined into 3 replicate groups, with each replicate group containing 2 fish. The fish were placed on ice, and blood samples were collected from the caudal vein using sterile disposable syringes. After centrifugation (4 °C, 4000 rpm, 10 min), the supernatant was collected and rapidly frozen in liquid nitrogen. The samples were then transferred and stored at -80 °C until further analysis. Following blood collection, the fish were dissected, and tissues (intestines, liver, mid-kidney, and muscles) were rinsed with a 0.9% saline solution to remove surface debris. The rinsed tissues were immediately placed in cryovials and flash-frozen in liquid nitrogen. Subsequently, the samples were transferred to -80 °C storage for further analysis.

Intraperitoneal injection test

The intraperitoneal injection experiment was conducted under the same experimental conditions, methods, and procedures as the oral administration experiment. The fish received the same dosage of amino acids as the oral experiment. The difference is that these five groups of fish received intraperitoneal injections instead of oral administration. The specific procedure for intraperitoneal injection was referenced from previous studies, with minor adjustments based on the specific species (Berge et al. 2002; Kinkel et al. 2010).

Determination of free amino acid content

To measure the levels of free amino acids in serum, a 0.5 mL of serum sample was mixed with 2.5 mL of a 4% salicylic acid solution. The mixture samples were then centrifuged at 14,000 rpm and 4 °C for 30 min. The supernatant was collecting for further analysis of free amino acids.

To measure the levels of free amino acids in tissues, an appropriate amount of animal tissue was weighed and placed in a glass homogenizer. The homogenizer was then placed in ice water, and the contents were homogenized for approximately 5 min. The contents were washed into a volumetric flask using physiological saline. A specific volume of an 8% salicylic acid solution was added to mix with the sample solution (tissue 20–50 mg / mL). After centrifugation (4 °C, 15,000 rpm, 10 min), the supernatant was collected for further analysis of free amino acids. The analysis of free amino acids was performed using a high-performance liquid chromatography system (1260 Infinity II, Agilent, USA) according to Matsushita (2010).

Determination of arginase and arginine synthetase activities

Fish were fasted for 24 h and anesthetized using MS-222 (150 mg / L, 5 min) followed by rapid dissection to isolate the intestinal canal, liver, kidney, muscle tissues. The arginase activity in the tissues were measured according to the method described by Wu (1996), with some modifications. Briefly, the mixture (0.15 mL) of arginase I was prepared consisting of 50 mM Tris-HCl buffer (pH 7.5), 3 mM MnCl2, 10 mM arginine (or citrulline), and cytoplasmic extract (0.2 to 0.5 mg protein). The enzyme and MnCl2 mixture were prepared before adding arginine (or citrulline). The final mixture was incubated at 26 °C for 20 min for reaction. At the end of the designated incubation time, 50 µL of 1.5 M HClO4 was added to terminate the reaction, and the neutralized solution was analyzed for ornithine content by using a high-performance liquid chromatography system.

Statistic analysis of data

Statistical analysis of the experimental data was performed using SPSS 22.0 software. One-way analysis of variance (ANOVA) was conducted to analyze the variance between different treatments. Duncan’s multiple range test was used to assess the degree of deviation between repeated measures. A significance level of 5% (P < 0.05) was considered statistically significant.

Results

Enzyme activity

The conversion rates of arginine to ornithine (arginase activity) were measured in various tissues of juvenile largemouth bass, as shown in Table 1. The results showed that the arginase activity in intestinal, liver, kidney and muscle tissues were 442, 420, 224, and 248 nmol / min / mL, respectively. Additionally, the conversion rates of citrulline to ornithine were measured in the liver and muscle tissues after 24 h post-feeding, and were found to be 77 nmol / min / mg protein and 65 nmol / min / mg protein, respectively.

Table 1 The rate of arginine or citrulline conversion to ornithine in various tissues of postprandial 24-hour juvenile largemouth bass (mean ± SD, n = 3)
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The effects of oral administration on serum free amino acids

In Fig. 1, it can be seen that oral administration of arginine or arginine-aspartate dipeptide significantly increased serum arginine levels in juvenile largemouth bass, from ~ 25 nmol/mL to ~ 55 nmol/mL after 10 min (P < 0.05). However, after 30 min, the levels returned to the baseline. The baseline serum ornithine levels in largemouth bass were around 95 nmol / mL. Oral intake of arginine or arginine-aspartate dipeptide significantly increased the serum ornithine levels (P < 0.05), reaching the highest levels after 1-hour post-administration (855 and 985 nmol / mL, respectively). Additionally, oral administration of arginine or arginine-aspartate dipeptide significantly increased the serum citrulline levels (P < 0.05), which increased from ~ 50 nmol / mL to around ~ 90 nmol / mL. Further, arginine-aspartate dipeptide significantly increased the serum aspartate levels (25–243 nmol / mL, P < 0.05), while citrulline significantly increased the concentrations of arginine (25–42 nmol / mL), ornithine (92–263 nmol / mL), citrulline (52-4959 nmol / mL), glutamate (43–68 nmol / mL), and aspartate (25–41 nmol / mL) in the serum (P < 0.05). Moreover, oral administration of glutamate significantly increased the levels of arginine (92–157 nmol / mL), citrulline (52–118 nmol / mL), aspartate (25–47 nmol / mL), and glutamate (43–367 nmol / mL) in the serum (P < 0.05).

Fig. 1
figure 1

Effects of oral administration of saline (A), arginine-aspartic dipeptide (B), arginine (C), citrulline (D), glutamate (E) on blood amino acid concentration of juvenile largemouth bass. Different letters indicate significant differences (P < 0.05). Values represent the average of three sets of data (each from a mixed sample of two fish). Sal saline, Arg arginine, Orn ornithine, Cit citrulline, Glu glutamate, Asp aspartate

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The effects of intraperitoneal injection of physiological saline on the levels of free amino acids in tissues

According to Fig. 2, intraperitoneal injection of physiological saline had no significant effect (P > 0.05) on the levels of arginine, ornithine, citrulline, and glutamate in the tissues of juvenile largemouth bass.

Fig. 2
figure 2

Effects of intraperitoneal injection of normal saline on the concentration of free amino acids in tissues of largemouth bass. (A) Serum, (B) Liver, (C) Kidney, (D) Muscle. Different letters indicate significant differences (P < 0.05). Values represent the average of three sets of data (each from a mixed sample of two fish). Sal saline, Arg arginine, Orn ornithine, Cit citrulline, Glu glutamate, Asp aspartate

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The effects of intraperitoneal injection of arginine or arginine-aspartate dipeptide on the levels of free amino acids in tissues

The effects of intraperitoneal injection of arginine or arginine-aspartate dipeptide on the levels of free amino acids in tissues are shown in Figs. 3 and 4. Serum arginine level in juvenile largemouth bass increased after intraperitoneal injection of arginine and arginine-aspartate dipeptide (both P < 0.05), reaching peak levels at 0.5-hour and 1-hour post-injection (3123 nmol / mL and 2562 nmol / mL, respectively). Similarly, intraperitoneal injection of arginine and arginine-aspartate dipeptide significantly raised the serum ornithine levels in largemouth bass after 1 h, elevating from baseline levels of ~ 90 nmol / mL to the peak level of ~ 4654 nmol / mL (P < 0.05). Additionally, the levels of arginine in the liver (0-739 nmol / g), kidney (27–600 nmol / g), and muscle tissues (185–4316 nmol / g) of largemouth bass were significantly increased after the intraperitoneal injection (P < 0.05). Similarly, the intraperitoneal injection raised the levels of ornithine in the liver (434-22515 nmol / g), kidney (794-114405 nmol / g), and muscle tissues (3409–21376 nmol / g, P < 0.05). Furthermore, the intraperitoneal injection of arginine and arginine-aspartate dipeptide also increased the concentrations of citrulline in the liver (70–200 nmol / g), kidney (49-1008 nmol / g), and muscle tissues (25–125 nmol / g, P < 0.05).

Fig. 3
figure 3

Effects of intraperitoneal injection of arginine on the concentration of free amino acids in tissues of largemouth bass. (A) Serum, (B) Liver, (C) Kidney, (D) Muscle. Different letters indicate significant differences (P < 0.05). Values represent the average of three sets of data (each from a mixed sample of two fish). Sal saline, Arg arginine, Orn ornithine, Cit citrulline, Glu glutamate, Asp aspartate

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Fig. 4
figure 4

Effects of intraperitoneal injection of arginine-aspartic dipeptide on the concentration of free amino acids in tissues of largemouth bass. (A) Serum, (B) Liver, (C) Kidney, (D) Muscle. Different letters indicate significant differences (P < 0.05). Values represent the average of three sets of data (each from a mixed sample of two fish). Sal saline, Arg arginine, Orn ornithine, Cit citrulline, Glu glutamate, Asp aspartate

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The effects of intraperitoneal injection of citrulline on the levels of free amino acids in tissues

The effects of intraperitoneal injection of citrulline on the levels of free amino acids in tissues are present in Fig. 5. Intraperitoneal injection of citrulline significantly increased the concentrations of arginine in the serum (25–44 nmol / mL), kidney (27–56 nmol / mL), and muscle (185–360 nmol / mL) of juvenile largemouth bass (P < 0.05). Furthermore, the administration of citrulline also significantly increased the ornithine concentration in the serum (92–418 nmol / mL), liver (434–1488 nmol / mL), kidney (794–3190 nmol / mL), and muscle (3409–6531 nmol / mL, P < 0.05). In addition, injecting citrulline led to significant increases in the levels of citrulline in the serum (51-6781 nmol / mL), muscle (25-6886 nmol / mL), and kidney (49-30036 nmol / mL), and liver (70-10989 nmol / mL) of largemouth bass (P < 0.05).

Fig. 5
figure 5

Effects of intraperitoneal injection of citrulline on the concentration of free amino acids in tissues of largemouth bass. (A) Serum, (B) Liver, (C) Kidney, (D) Muscle. Different letters indicate significant differences (P < 0.05). Values represent the average of three sets of data (each from a mixed sample of two fish). Sal saline, Arg arginine, Orn ornithine, Cit citrulline, Glu glutamate, Asp aspartate

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The effects of intraperitoneal injection of glutamate on the levels of free amino acids in tissues

The effects of intraperitoneal injection of glutamate on the levels of free amino acids in tissues are present in Fig. 6. Intraperitoneal injection of glutamate caused significant increases in the concentrations of arginine in serum (25–41 nmol / mL) and kidney (27–67 nmol / mL) of juvenile largemouth bass (P < 0.05). The intraperitoneal injection of glutamate also led to a significant elevation in the levels of ornithine in the serum (92–114 nmol / mL) and muscle (3409–6596 nmol / mL) of largemouth bass (P < 0.05). Additionally, the concentrations of citrulline in the serum (51–97 nmol / mL), liver (70–919 nmol / mL), kidney (49-1009 nmol / mL), and muscle (25-1623 nmol / mL) of largemouth bass were notably affected by the intraperitoneal injection of glutamate (P < 0.05). Moreover, the levels of glutamate in the serum (43-2621 nmol / mL), liver (7053–14172 nmol / mL), kidney (8515–21061 nmol / mL), and muscle (600–7636 nmol / mL) of largemouth bass were significantly impacted by glutamate administration (P < 0.05).

Fig. 6
figure 6

Effects of intraperitoneal injection of glutamate on the concentration of free amino acids in tissues of largemouth bass. (A) Serum, (B) Liver, (C) Kidney, (D) Muscle. Different letters indicate significant differences (P < 0.05). Values represent the average of three sets of data (each from a mixed sample of two fish). Sal saline, Arg arginine, Orn ornithine, Cit citrulline, Glu glutamate, Asp aspartate

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Discussion

The arginine family amino acid concentrations in tissues

In this experiment, the serum arginine concentration in largemouth bass was measured after 24 h of fasting. The concentration was found to be approximately 25 nmol / mL, which was consistent with the results of a previous study by Li et al. (2020a). Among all the arginine family amino acids, the concentration of arginine in the serum of juvenile largemouth bass is the lowest (Fig. 1A). It is worth noting that previous studies on different fish species have consistently found higher levels of arginine in their blood compared to the level in largemouth bass. For example, the reported values were 70–200 nmol / mL in channel catfish (Pohlenz et al. 2013), 50–60 nmol / mL in turbot (Scophthalmus maximus L.) (Xu et al. 2016), 160 nmol / mL in hybrid tilapia (Li et al. 2021), and 200–300 nmol / mL in rainbow trout (Oncorhynchus mykiss) (Barrows et al. 2007).

Muscle tissue of most fish accounts for more than 50% of total body mass (Karl and Bastrop 1995), which also comprise the largest reservoir of free amino acids in body (Buentello and Gatlin 2001). In this study, the concentration of ornithine in largemouth bass muscle after fasting 24 h was significantly higher than that of other arginine family amino acids (Fig. 2D), which is similar to the results observed in mice (Marini et al. 2010). Ornithine is considered a non-proteogenic amino acid and an intermediate in the biosynthesis of L-arginine (Eberhardt et al. 2014). Marini et al. (2010) proposed a decrease in available arginine reserves due to high ornithine flux from arginine hydrolysis in mice. Thus, ornithine flux can be regarded as an alternative measure of arginine availability (Marini et al. 2010). In this study, we observed high arginase activity in the intestines, liver, kidney, and muscle of largemouth bass (Tables 1 and 224–442 nmol / min / mg protein), which is consistent with the results reported by Li et al. (2021) and Kong et al. (1998). In contrast, the intestinal arginase activity in weaned piglets was only 32 nmol / min / mg protein (Wu et al. 1996). The hepatic arginase activity of orange-spotted grouper (Epinephelus coioides) (Han et al. 2018) and sea bass (Dicentrarchus labrax) (Tulli et al. 2007) was also only 6–9 and 19.72 nmol / min / mg protein, respectively. Therefore, significantly high activity of arginase is responsible for the elevated ornithine and reduced arginine flux in largemouth bass.

Effects of oral and intraperitoneal administration of arginine or arginine-aspartate dipeptide on metabolism

In a previous study, it was found that largemouth bass did not experience any increase in serum arginine concentration at 2 h after consuming high levels of arginine (Li et al. 2021). In the present study, it was observed that the serum arginine concentration increased rapidly in largemouth bass after they were given an oral administration of arginine or arginine-aspartate dipeptide. The concentration reached its peak at approximately 10 min and then falling back at 30 min (Fig. 1B). This indicates that the dipeptide form and free arginine can be absorbed through the intestine of largemouth bass. However, the absorbed arginine was further rapidly hydrolyzed in the tissues of this species. Therefore, the serum arginine concentration has returned to baseline value in samples collected 2 h after a meal in largemouth bass (Li et al. 2021).

The concentration of ornithine rapidly increased from the initial level of 92 nmol / mL after oral administration, reaching a peak of about 900 nmol / mL after an hour. In contrast, other amino acids remained stable. However, the concentration of arginine increased only slightly 10 min after oral administration, but not as much as ornithine. These results indicate that arginine is converted into ornithine in the tissues of largemouth bass. These findings are consistent with previous studies by Li et al. (2020a), 2021) on largemouth bass. However, in other teleost fish, postprandial blood arginine concentrations were consistently positively correlated with dietary arginine levels (Chiu et al. 1986; Fauzi et al. 2019; Pohlenz et al. 2013; Walton and Wilson 1986). Therefore, there are significant variations in arginine metabolism among different species.

The intestine is a metabolically active organ that extensively metabolizes amino acids during absorption (first-pass metabolism; Li et al. 2021). To eliminate the influence of the gastrointestinal tract, we administered amino acids through intraperitoneal injection. This study found that intraperitoneal injection of arginine significantly affected the concentrations of arginine and ornithine in the bloodstream of largemouth bass (Figs. 3A and 4A). The increase of serum ornithine levels was also much higher than that of arginine. When arginine was substituted with arginine-aspartate dipeptide, the peak value of arginine was delayed from half an hour to one-hour post-injection. Besides, no significantly difference was observed in ornithine production between arginine and arginine-aspartate dipeptide treatments. This suggests that the structure of the dipeptide may have a positive effect on protecting arginine from degradation but very limited. A significant increase in serum aspartate and glutamate levels also indicates that the dipeptide is rapidly broken down in tissues. There is a good conversion between aspartic acid and glutamate. This phenomenon can be explained by higher aminotransferase activity in fish tissues (Li et al. 2020b). Overall, even when bypassing the intestine and entering directly into the bloodstream, arginine is still rapidly degraded into ornithine. These findings are consistent with the studies conducted by Li et al. (2021) and Kong et al. (1998). Furthermore, several studies have revealed that changes in free arginine concentration affect the activity of arginase in fish liver. Tulli et al. (2007) found a positive correlation between the arginase activity in the liver of sea bass and the dietary arginine levels. Han et al. (2018) have verified that high levels of dietary arginine significantly increased the activity of hepatic arginase in orange-spotted grouper. Therefore, the increase of arginine in tissues after intraperitoneal injection may improve the arginase activity in the liver and kidney of largemouth bass.

This study also showed that arginine intraperitoneal injection results in massive production and accumulation of glutamate acid in the muscle and kidney. In mammals, ornithine aminotransferase (OAT) is responsible for converting ornithine into P5C and glutamate (Ginguay et al. 2017; Wekell and Brown Jr 1973). Present results indicate that OAT activity is present in the muscle and kidney of largemouth bass, resulting in a glutamate production at high ornithine concentrations. An increases of tissue citrulline were observed in the arginine intraperitoneal injection group, which suggests that ornithine transcarbamylase (OTC) may also be present. A review based on mammalian studies describes the cycling of citrulline between organs. In the intestine, arginine is synthesized into ornithine by arginase and further synthesized into citrulline by OTC (Cynober et al. 2010). There is growing evidence that there is a urea cycle in fish under specific growth stages or environmental conditions (Wang et al. 2021). For example, rainbow trout exhibit higher CPSIII and OAT activities during early developmental stages (Wright et al. 1995).

Effects of oral and intraperitoneal administration of citrulline on metabolism

In this study, oral administration or intraperitoneal injection of citrulline led to a rapidly and significantly increased serum and tissue levels of citrulline. Within one hour of oral administration, blood citrulline levels increased from 52 nmol / mL to 5000 nmol / mL, indicating that citrulline was effectively absorbed by the intestine. After intraperitoneal injection of citrulline, substantial amounts of free citrulline accumulated in various tissues and was maintained at high levels for up to 4 h. This suggests that the metabolic turnover rate of citrulline is lower in these tissues. Currently, arginine synthesis is the only metabolic fate of citrulline in animals, which partially explains the substantial accumulation of free citrulline in tissues (Marini et al. 2010). Additionally, both serum arginine and ornithine levels showed moderate increase after intraperitoneal injection or oral administration of citrulline, suggesting that citrulline may be converted into arginine and further metabolized into ornithine. In mammals, the metabolic cycling of citrulline between organs was clearly described by Cynober et al. (2010). Briefly, citrulline produces arginine in the kidney under the action of argininosuccinate synthase and argininosuccinate lyase, which in turn produces ornithine and citrulline in the intestine. The results of the present experiments suggest that under specific conditions, citrulline synthesis or urea cycle metabolic mechanisms may exist in fish (Clark et al. 2020a; Wang et al. 2021). Recently, it has been confirmed that rainbow trout can synthesize arginine endogenously through dietary supplementation with citrulline (Clark et al. 2020b). Furthermore, another study on rainbow trout also showed that the addition of citrulline to the diet significantly increases the levels of arginine in the plasma and muscle tissue (Chiu et al. 1986; Clark et al. 2020a, b).

As mentioned before, the only fate of citrulline in vivo is conversion into arginine. In rainbow trout, citrulline is more effective than arginine in maintaining high blood concentrations of arginine (Lassala et al. 2009; Clark et al. 2020a). However, it was found in this experiment that oral and intraperitoneal administration of citrulline increased the concentration of ornithine rather than arginine. Present experiment also showed that oral and intraperitoneal administration of arginine could be rapidly metabolized to ornithine. No significant increase in arginine levels was detected in various tissues of largemouth bass with 10 mM citrulline substrate (Table 1). Instead, the results showed a corresponding increase in ornithine in the liver and muscle (Table 1). Therefore, it is possible that a certain amount of arginine synthesis occurred under short-term injection with high concentrations of citrulline, but most of the arginine was rapidly degraded.

The accumulation of large amounts of citrulline in the body may regulate metabolic balance, resulting in changes in the levels of related free amino acids (e.g., glutamate and aspartate). The exact process of arginine synthesis from citrulline requires further verification. The administration of arginine or arginine-aspartate dipeptide led to an accumulation of glutamate in the kidneys and muscles. However, intraperitoneal injection of citrulline does not have the same effect. Although citrulline administration led to an increase of ornithine (nearly 3000 nmol / mL) in tissues, it was much lower than the accumulation values caused by direct intraperitoneal injection of arginine or arginine-aspartate dipeptide (> 8000 nmol / mL). These results indicate that OAT-regulated transamination may only occur under very high concentrations of ornithine substrates.

Effects of oral and intraperitoneal administration of glutamate on metabolism

In mammals, enterocytes can convert glutamate, glutamine, and proline into citrulline. The citrulline can then converted into arginine by the kidney as illustrated in Fig. 7 (Flynn and Wu 1996; O’Sullivan et al. 1998; Wu et al. 1997). However, there is limited study on arginine synthesis in fish. According to reports, the enzyme activity involved in arginine biosynthesis in fish is relatively low, such as P5CS, OAT, and CPS III (Andersen et al. 2016). In the present study, administrating glutamate resulted in a significant increase in serum citrulline concentration, indicating the possibility of citrulline synthesis from glutamate. Besides, intraperitoneal injection of glutamate significantly increased arginine concentrations in serum and kidneys. All these results indicated that the synthesis of arginine from glutamic acid may exist in this species. However, in vitro experiments, any citrulline and arginine formation did not observe in the intestines, liver, kidneys and skeletal muscles of hybrid tilapia, largemouth bass and zebrafish, using 2 mM glutamate, glutamine, or proline substrates, respectively (Li et al. 2020b). The lack of synthesis may be due to the coordinated action of multiple enzymes (e.g., P5C, OAT, and CPS III) required in different tissues for arginine biosynthesis. Notably, the absence of arginine synthesis in various tissues of largemouth bass in vitro may be due to the presence of highly active arginase, which may have prevented the retention of any synthesized arginine. In present study, it has been observed that the citrulline accumulation is highest in skeletal muscle after intraperitoneal injection of glutamate (Fig. 6). This indicates the muscle of largemouth bass is capable of synthesizing citrulline under high concentrations of glutamate. In rainbow trout, key enzymes responsible for endogenous arginine synthesis were detected only in muscles (Felskie et al. 1998; Korte et al. 1997; Todgham et al. 2001). Further research is needed to identify the functional organs responsible for citrulline and arginine synthesis in largemouth bass.

Fig. 7
figure 7

Arginine metabolism in mammals

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The arginine requirements in fish have been extensively studied. In a previous study by Chiu et al. (1986), when equal amounts of citrulline, arginine, glutamic acid and ornithine were added to feed, it was found that citrulline could replace same amount of arginine. However, ornithine and glutamate could not replace the same amount of arginine. In that study, Chiu et al. (1986) did not set a control group with amino acid that is not belong arginine-family. Therefore, although the results showed that the substitution effect of glutamic acid for arginine is inferior to than citrulline and ornithine, this does not deny the possibility of endogenous synthesis of arginine from glutamic acid. Buentello and Gatlin (2000) demonstrated that the growth efficiency, feed efficiency and plasma arginine concentration in channel catfish was significantly improved by adding glutamic acid to feed compared with glycine. Therefore, some teleost fish may have the ability to synthesize arginine from glutamic acid through intermediate conversion of citrulline, such as largemouth bass, rainbow trout, and channel catfish. However, arginine can also be rapidly metabolized and degraded in some fish species. Glutamate, ornithine, and citrulline can affect the endogenous synthesis of arginine to varying degrees, thereby influencing the dietary arginine requirement in these fish. It is necessary to further investigate and understand the arginine requirements and metabolism in different fish species to improve the utilization of dietary protein and promote growth.

Conclusion

In summary, the presence of arginase in the intestines, liver, kidney, and muscle results in a prolonged stable circulation of low arginine levels in largemouth bass (Micropterus salmoides). Largemouth bass may have a capability that synthesizes citrulline from glutamate and then converts citrulline to arginine, which mechanism may differ from mammalian. This study provides valuable insights into the metabolism of arginine in largemouth bass and highlights the importance of understanding the biochemical pathways involved in amino acid metabolism in fish. These findings have significant implications for the development of aquaculture practices and for advancing our understanding of fish nutrition and physiology.