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. 2020 May 16;98(6):skaa164. doi: 10.1093/jas/skaa164

Ruminal microbes of adult sheep do not degrade extracellular l-citrulline

Kyler R Gilbreath 1, Fuller W Bazer 1, M Carey Satterfield 1, Jason J Cleere 1, Guoyao Wu 1,✉
PMCID: PMC7344112  PMID: 32415842

Abstract

This study determined whether extracellular citrulline is degraded by ruminal bacteria of sheep. In the first experiment, whole rumen fluid (3 mL) from six adult Suffolk sheep was incubated at 37 °C with 5 mM l-glutamine (Gln), l-glutamate (Glu), l-arginine (Arg), or l-citrulline (Cit) for 0, 0.5, 1, and 2 h or with 0, 0.5, 2, or 5 mM Gln, Glu, Arg, or Cit for 2 h. An aliquot (50 µL) of the incubation solution was collected at the predetermined time points for amino acids (AA) analyses. Results showed extensive hydrolysis of Gln into Glu and ammonia, of Arg into l-ornithine and l-proline, but little or no degradation of extracellular Cit or Glu by ruminal microbes. In the second experiment, six adult Suffolk sheep were individually fed each of three separate supplements (8 g Gln , Cit, or urea) on three separate days along with regular feed (800 g/animal). Blood (2 mL) was sampled from the jugular vein prior to feeding (time 0) and at 0.5, 1, 2, and 4 h after consuming the supplement. Plasma was analyzed for AA, glucose, ammonia, and urea. The concentrations of Cit in the plasma of sheep consuming this AA increased (P < 0.001) by 117% at 4 h and those of Arg increased by 23% at 4 h, compared with the baseline values. Urea or Gln feeding did not affect (P > 0.05) the concentrations of Cit or Arg in plasma. These results indicate that Cit is not metabolized by ruminal microbes of sheep and is, therefore, absorbed as such by the small intestine and used for the synthesis of Arg by extrahepatic tissues.

Keywords: amino acids, arginine, degradation, ruminal bacteria

Introduction

There is growing interest in the nutritional and physiological roles of amino acids (AA) in growth, development, and survival of livestock, and one of the AA is l-arginine (Arg; Wu, 2018; Reynolds et al., 2019; Sciascia et al., 2019). In the rumen of ruminants, AA are degraded to ammonia and their carbon skeletons by bacterial enzymes, including deaminases, transaminases, hydrolases, and decarboxylases (Wu, 2013). In the presence of α-ketoacids (e.g., pyruvate, oxaloacetate, and α-ketoglutarate that are products of carbohydrate metabolism) and sulfur, AA-derived ammonia is utilized by ruminal bacteria for the synthesis of new AA and proteins (Owens and Bergen, 1983; Firkins et al., 2007). The rates of ruminal AA catabolism are high, such that proteinogenic free AA in diets do not escape the rumen (Lewis, 1955; Annison, 1956; Chalupa, 1975, 1976; Butter and Folds, 1985; Cozzi et al., 1995; Bach et al., 2005). Thus, the microbial population within the rumen has long been considered to have the capability to extensively degrade all dietary AA (Lewis and Emery, 1962; Chalupa, 1976; Kung and Rode, 1996; NRC, 2000, 2001). For this reason, rumen-protected AA (e.g., Arg) are used as supplements in diets for ruminants (e.g., Peine et al., 2018; Teixeira et al., 2019; Zhang et al., 2019a). Recently, we reported that extracellular Cit was not catabolized by ruminal microbes of steers (Gilbreath et al., 2019, 2020). However, the degradability of extracellular l-citrulline (Cit, the immediate precursor of Arg) by ovine ruminal microbes is not known. Therefore, in the present study, an in vitro experiment was conducted with ruminal fluid of adult sheep to address this important question, with Arg, l-glutamine (Gln), and l-glutamate (Glu) as reference AA. In addition, an in vivo experiment was performed to determine whether oral administration of Cit resulted in increases in concentrations of Cit and Arg in plasma of adult sheep.

Materials and Methods

Adult Sulfolk female sheep (60 to 65 kg) used in the experiments were housed individually in the Animal Science Teaching, Research, and Extension Center of Texas A&M University. The animals had free access to a soybean hulls-, wheat middlings-, and corn-based diet (Satterfield et al., 2013) as well as water. The diet contained 9.8% water, 12.8% crude protein, 5.36% crude fat, 39.8% neutral detergent fiber (NDF), 29.2% non-NDF (nonfiber) carbohydrates (carbohydrates soluble in neutral detergent solution), 3.0% minerals, 0.04% vitamins, and gross energy of 4,030 kcal/kg on an as-fed basis (90.2% dry matter). The content of AA in the diet (%; as-fed basis) was: alanine, 0.84; arginine, 0.76; asparagine, 0.66; aspartate, 0.75; cysteine, 0.24; glutamate, 1.01; glutamine 1.16; glycine, 0.63; histidine, 0.28; isoleucine, 0.54; leucine, 1.07; lysine, 0.64; methionine, 0.21; phenylalanine, 0.63; proline, 1.02; serine, 0.65; threonine, 0.49; tryptophan, 0.16; tyrosine, 0.48; and valine, 0.64. No Cit was detected in the diet. This study was approved by the Institutional Animal Care and Use Committee of Texas A&M University. The research protocol followed the U. S. Department of Agriculture Animal Welfare Regulations.

In vitro experiment

Fresh ruminal fluid was collected from six adult sheep that had been deprived of food for 16 h. Specifically, sheep were stunned using a captive bolt gun followed immediately by exsanguination (Satterfield et al., 2013). Immediately upon exsanguination, ruminal fluid (50 mL) was taken from each animal and gently mixed. The sample was then aliquoted into polypropylene tubes (3 mL/tube) to determine time- and concentration-dependent changes in AA metabolism. The tubes contained one of the following: no addition of AA, 2.2 mg Gln (5 mM), 2.2 mg Glu (5 mM), 3.1 mg Arg-HCl (5 mM), or 2.7 mg Cit (5 mM). No carbohydrate or sulfur compounds were added to the incubation medium. The initial pH of the unsupplemented ruminal fluid was 6.64, whereas the initial pH of the ruminal fluid with the addition of 5 mM Gln (a neutral AA), 5 mM Arg-HCl (a neutralized form of Arg), and 5 mM Glu was 6.64, 6.64, and 6.61, respectively. These pH values were within the normal ranges (6.4 to 6.8) for the ruminal fluid of adult sheep (Jasmin et al., 2011). Although Glu is an acidic AA at ruminal pH, its addition to the ovine ruminal fluid had little effect on the pH of the fluid. This is because: 1) the concentration of the added Glu (5 mM) is relatively low compared with those of total short-chain fatty acids (77 to 97 mM; much stronger acids than Glu) in the ruminal fluid of healthy sheep (Gaebel et al., 1987) and 2) the ruminal fluid of healthy sheep contains other organic acids, such as lactic acid (a much stronger acid than Glu) and branched-chain fatty acids at 0.2 to 2 mM (Braun et al., 1992; Hobson and Stewart, 1997). No compounds or reagents were added to the ruminal fluid to adjust the initial pH of the incubation medium. The tubes were gassed with CO2 for 20 s, capped, and then incubated (70 oscillations/min) at 37 °C. At 0, 0.5, 1, and 2 h of the incubation period, an aliquot (50 µL) of the incubation solution was collected from each tube.

To determine concentration-dependent changes in AA catabolism, a 5 mM AA solution was diluted with ruminal fluid to prepare 0.5 and 2 mM solutions. In the “0 mM” tube, no AA was added to the ruminal fluid. These sets of tubes were run simultaneously with those for assessing time-dependent changes in AA catabolism as described previously. No carbohydrate or sulfur compounds were added to the incubation medium. The solutions (in polypropylene tubes) were gassed with CO2 for 20 s, capped, and then incubated (70 oscillations/min) at 37 °C. At the end of a 2-h incubation period, an aliquot (50 µL) of the incubation solution was collected from the tube.

For AA analysis, an aliquot (50 µL) of 1.5 M HClO4 was added to the collected incubation sample, followed by the addition of 10 µL of 2 M K2CO3 for neutralization and then 150 µL of high-performance liquid chromatography (HPLC)-grade water. The extracts were stored at −20 °C until analyzed for AA by HPLC (Wu and Meininger, 2008) and for urea using urease and Glu dehydrogenase (Wu, 1995). In the latter assay, urea was hydrolyzed by urease to ammonia, which reacted with α-ketoglutarate to form Glu by the coupling nicotinamide adenine dinucleotide (reduced)-dependent Glu dehydrogenase.

To determine the uptake of AA by ruminal microbes, ruminal fluid (1 mL; containing microorganisms) was incubated as described above, except that the medium contained 5 mM l-[U-14C]Glu, l-[U-14C]Cit, l-[U-14C]Arg, or l-[U-14C]Gln (100 dpm/nmol) for 2 h. The tracers were purchased from American Radiolabeled Chemicals Inc. (St. Louis, MO) and purified before use (Chen et al., 2009). Blanks, in which Krebs-bicarbonate buffer replaced ruminal fluid, were included for parallel incubations. At the end of the incubation period, 0.2 mL of 1.5 M HClO4 was added through the stopper into the incubation medium and 14CO2 was collected into Soluene, as we described previously (Li et al., 2016). The radioactivity of 14CO2 was determined by a liquid scintillation counter (Zhang et al. 2019b). Thereafter, the acidified medium was neutralized with 0.1 mL of 2 M K2CO3 as described above. An aliquot (100 µL of the whole extract) was analyzed for separation and collection of AA fractions using HPLC (Wu et al., 2000), as well as for 14C-urea using urease on the basis of 14CO2 production (Wu and Brosnan, 1992). Incorporation of 14C-AA into microbial proteins was determined by measuring the radioactivity of 14C-labeled proteins, as we described previously (Dai et al., 2010). The sum of labeled products was used to calculate the uptake of each radiolabeled substrate.

In vivo experiment

Six adult sheep were individually fed each of three supplements (Cit, Gln, or urea) on three separate days, with 2 d apart between supplements. These animals were not the same as those used for the in vitro experiment. Each supplement (8 g) was offered along with 800 g of the soybean hulls-, wheat middlings-, and corn-based diet (Satterfield et al., 2013) to each sheep. The composition of nutrients in this diet was the same as described previously. The supplemental amount of Cit and Gln (~1% of the diet) was chosen on the basis of the findings of our previous studies that dietary supplementation with 1% Cit (Wu, G., unpublished work) or 1% Gln (Wu et al., 2011) to adult swine increased the plasma concentrations of Cit and Gln by 127% and 30%, respectively. The supplemental amount of urea (~1% of the diet) was chosen because this dose had been reported to be safe for adult sheep (Zhang et al., 2016b). Blood (2 mL) was sampled from the jugular vein of the sheep into heparinized tubes prior to feeding (time 0) and at 0.5, 1, 2, and 4 h after consuming the supplement (8 g) plus the 800 g feed. It took each sheep about 10 min to eat all the supplements plus the feed. We chose a 4-h period of blood sampling based on the results of our preliminary experiment that the concentrations of Arg, Cit, and Gln at 0 h did not differ (P > 0.05) from those at 6 h after oral administration of 8 g Arg, Cit, or Gln to adult sheep (Gilbreath, K. R. and G. Wu, unpublished work). Blood samples (1 mL) were centrifuged immediately to obtain plasma. An aliquot (100 µL) of plasma was acidified with an equal volume of 1.5 M HClO4, followed by the addition of 50 µL of 2 M K2CO3 for neutralization and then 2.25 mL of HPLC-grade water. The extracts were stored at −20 °C until analyzed for AA by HPLC (Wu and Meininger, 2008). Ammonia, urea, and glucose were analyzed by using enzymatic methods, as we described previously (Satterfield et al., 2012, 2013). The intra-assay variations (expressed as coefficient of variation [standard deviation/mean]) to monitor the deviation within the same assay) of ammonia, urea, and glucose were 1.5%, 1.4%, and 1.2%, respectively. The inter-assay variations (expressed as coefficient of variation to monitor the deviation between the assays) of ammonia, urea, and glucose were 1.8%, 1.6%, and 1.3%, respectively.

Statistical analysis

Data were analyzed by one-way analysis of variance for repeated measures data (Lee et al., 2019) or one-way ANOVA, as indicated in tables (Assaad et al., 2014). Log transformation of variables was performed when the variance of data were not homogenous, as assessed by the Levene’s test (Wei et al., 2012). Differences among treatment means were determined using the Student–Newman–Keuls multiple comparison test. P-values ≤ 0.05 were taken to indicate statistical significance.

Results

In vitro experiment

During a 2-h period of incubation, concentrations of Gln and Arg in the medium decreased (P < 0.05) and those of Asp, Ala, and ornithine increased (P < 0.05) with time, but those of Glu, Cit, and ammonia did not differ (P > 0.05) among different time points (Tables 1–3). The concentrations of Ser in the ruminal fluid incubated with 5 mM Gln increased (P < 0.05) gradually with time (Table 2). In the ruminal fluid with the added 5 mM Gln and 5 mM Arg, their concentrations at the end of the 2-h period of incubation were decreased (P < 0.001) by 38% and 26%, respectively (Table 1). Increasing the extracellular concentrations of Gln or Arg from 0.5 to 5 mM increased (P < 0.001) their utilization by ruminal microbes in a dose-dependent manner (Table 2). The major products of Gln were Glu, ammonia, Ala, Ser, and Asp, whereas the major products of Arg were ornithine, Pro, and ammonia (Tables 1–3). In contrast, there was no detectable catabolism of extracellular Cit or Glu by ruminal microbes during a 2-h period of incubation.

Table 1.

Time-dependent changes of AA concentrations in the incubated ruminal fluid of sheep after the addition of an AA1

Addition to medium Hour Asp Glu Gln Cit Arg Orn Ala Ammonia2
None 0 22.6 ± 1.8d 72.8 ± 5.0 49.3 ± 4.6a 11.5 ± 0.93 30.7 ± 2.6a 31.2 ± 3.3b 16.7 ± 1.3d 1.05 ± 0.10
0.5 25.4 ± 2.1c 74.5 ± 5.5 47.5 ± 4.9a 10.8 ± 0.95 28.1 ± 2.4ab 32.7 ± 3.5b 19.4 ± 1.5c 1.07 ± 0.12
1 29.7 ± 2.0b 80.6 ± 6.3 41.3 ± 4.2b 10.4 ± 0.90 26.5 ± 2.1bc 34.6 ± 3.6ab 25.2 ± 1.6b 1.02 ± 0.13
2 34.2 ± 2.2a 84.2 ± 6.9 35.6 ± 3.4c 11.0 ± 0.98 24.0 ± 1.9c 37.1 ± 3.6a 31.7 ± 2.6a 1.04 ± 0.13
P-value <0.001 0.275 <0.001 0.884 0.021 0.033 <0.001 0.926
5 mM Gln 0 23.0 ± 2.0d 73.1 ± 5.6d 5,041 ± 12a 11.8 ± 1.1b 30.6 ± 2.8a 30.9 ± 3.1c 16.9 ± 1.4d 1.07 ± 0.10d
0.5 27.7 ± 1.9c 937 ± 42c 4,133 ± 10b 12.4 ± 1.0b 28.9 ± 2.7ab 33.2 ± 3.0bc 30.3 ± 1.9c 1.98 ± 0.16c
1 31.5 ± 2.3b 1,253 ± 48b 3,812 ± 24c 13.7 ± 1.2ab 26.3 ± 2.3bc 36.0 ± 3.3ab 44.2 ± 5.0b 2.33 ± 0.20b
2 39.2 ± 2.7a 1,741 ± 72a 3,146 ± 39d 15.9 ± 1.3a 24.5 ± 2.2c 43.8 ± 3.7a 70.8 ± 7.4a 2.95 ± 0.24a
P-value <0.001 <0.001 <0.001 0.037 0.023 <0.001 <0.001 <0.001
5 mM Cit 0 22.8 ± 1.9d 72.4 ± 5.2 49.8 ± 4.7a 5,018 ± 13 30.1 ± 2.7a 31.5 ± 3.2b 16.5 ± 1.2d 1.08 ± 0.13
0.5 25.8 ± 2.0c 75.1 ± 5.8 47.2 ± 4.6a 5,026 ± 15 27.9 ± 2.6ab 32.3 ± 3.4b 19.2 ± 1.6c 1.13 ± 0.12
1 30.1 ± 2.2b 77.8 ± 6.6 41.5 ± 4.1b 5,012 ± 11 26.3 ± 2.3bc 34.9 ± 3.5ab 25.6 ± 1.7b 1.06 ± 0.14
2 34.5 ± 2.3a 83.9 ± 7.2 36.0 ± 3.5c 5,006 ± 16 23.8 ± 2.5c 37.6 ± 3.9a 31.9 ± 2.1a 1.03 ± 0.12
P-value <0.001 0.308 <0.001 0.985 0.019 0.037 <0.001 0.938
5 mM Glu 0 22.4 ± 1.8d 5,065 ± 28 49.6 ± 4.5a 12.0 ± 1.0 30.8 ± 2.5a 31.0 ± 3.1b 16.2 ± 1.2d 1.03 ± 0.12
0.5 26.0 ± 2.1c 5,070 ± 33 47.3 ± 4.4a 11.6 ± 1.1 28.5 ± 2.4ab 32.5 ± 3.2b 19.1 ± 1.3c 1.04 ± 0.11
1 30.3 ± 2.4b 5,059 ± 35 41.8 ± 4.1b 11.3 ± 0.97 26.0 ± 2.6bc 34.8 ± 3.3ab 25.0 ± 1.8b 1.08 ± 0.12
2 34.7 ± 2.6a 5,063 ± 37 35.2 ± 3.6c 11.5 ± 1.2 24.9 ± 2.3c 37.6 ± 3.5a 31.3 ± 2.0a 1.10 ± 0.14
P-value <0.001 0.962 <0.001 0.852 0.017 0.028 <0.001 0.951
5 mM Arg `0 22.5 ± 2.1d 73.5 ± 6.0 49.7 ± 5.3a 11.3 ± 1.2d 5,027 ± 42a 31.7 ± 3.3d 16.4 ± 1.4d 1.06 ± 0.12d
0.5 27.3 ± 2.3c 76.2 ± 6.1 47.0 ± 5.0a 40.6 ± 3.7c 4,513 ± 38b 72.6 ± 4.0c 19.5 ± 1.6c 2.11 ± 0.23c
1 32.6 ± 2.5b 76.8 ± 7.3 42.3 ± 4.6b 67.0 ± 5.1b 4,306 ± 36c 95.4 ± 6.1b 24.7 ± 1.9b 2.48 ± 0.27b
2 36.5 ± 2.7a 77.5 ± 6.9 36.4 ± 3.8c 109 ± 7.4a 3,728 ± 31d 142 ± 8.7a 32.2 ± 2.1a 3.60 ± 0.39a
P-value <0.001 0.325 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
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1Values, expressed as nmol/ml of ruminal fluid for AA and as µmol/mL of ruminal fluid for ammonia, are means ± SEM, n = 6.

2NH3 plus NH4+.

a–dWithin a column, means not sharing the same superscript differ (P < 0.05), as analyzed by one-way ANOVA for repeated measure data, followed by the Student–Newman–Keuls multiple comparison test.

Table 3.

Concentrations of alanine and proline in the incubated ovine ruminal fluid after addition of various concentrations of an AA1

Addition to medium Concentrations, mM Ala Pro
Gln 0 31.7 ± 2.6d 29.4 ± 3.0
0.5 42.5 ± 4.8c 30.6 ± 3.2
2 56.0 ± 6.3b 31.2 ± 3.2
5 70.8 ± 7.4a 31.8 ± 3.3
P-value <0.001 0.896
Cit 0 31.7 ± 2.6 29.4 ± 3.0
0.5 30.5 ± 2.8 31.1 ± 3.4
2 31.9 ± 3.1 30.9 ± 3.3
5 32.3 ± 3.0 30.3 ± 3.5
P-value 0.912 0.904
Glu 0 31.7 ± 2.6 29.4 ± 3.0
0.5 32.4 ± 3.0 29.8 ± 3.2
2 33.1 ± 2.9 30.1 ± 3.7
5 33.9 ± 2.8 30.7 ± 3.6
P-value 0.878 0.973
Arg 0 31.7 ± 2.6 29.4 ± 3.0d
0.5 32.0 ± 3.0 418 ± 55c
2 32.6 ± 2.7 695 ± 77b
5 33.4 ± 3.1 863 ± 96a
P-value 0.980 <0.001
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1Rumen fluid was incubated for 2 h in the presence of an added AA. These sets of tubes were run simultaneously with those described in Table 1. Values, expressed as nmol/mL of ruminal fluid for AA, are means ± SEM, n = 6.

a–dWithin a column, means not sharing the same superscript differ (P < 0.05), as analyzed by one-way ANOVA, followed by the Student–Newman–Keuls multiple comparison test.

Table 2.

AA profile in the incubated ruminal fluid of sheep after addition of various concentrations of an AA1

Addition to medium Concentrations, mM Asp Glu Gln Cit Arg Orn Ser Ammonia2
Gln 0 34.2 ± 2.2b 84.2 ± 6.9d 35.6 ± 3.4d 11.0 ± 0.98b 24.0 ± 1.9 37.1 ± 3.6c 31.6 ± 1.8c 1.04 ± 0.13d
0.5 36.0 ± 2.4ab 336 ± 17c 284 ± 22c 12.8 ± 0.96b 24.4 ± 2.1 39.6 ± 3.5bc 33.4 ± 2.0bc 1.28 ± 0.15c
2 37.8 ± 2.4ab 720 ± 38b 1,402 ± 31b 13.6 ± 1.1ab 23.9 ± 2.5 41.0 ± 3.8ab 35.1 ± 2.1ab 1.70 ± 0.19b
5 39.2 ± 2.7a 1,741 ± 72a 3,146 ± 39a 15.9 ± 1.3a 24.5 ± 2.2 43.8 ± 3.7a 37.8 ± 2.2a 2.66 ± 0.25a
P-value <0.001 <0.001 <0.001 0.019 0.995 0.041 <0.001 <0.001
Cit 0 34.2 ± 2.2 84.2 ± 6.9 35.6 ± 3.4 11.0 ± 0.98c 24.0 ± 1.9 37.1 ± 3.6 31.6 ± 1.8 1.04 ± 0.13
0.5 33.8 ± 2.7 82.7 ± 7.1 36.3 ± 3.2 512 ± 16b 25.3 ± 2.4 38.6 ± 4.0 31.3 ± 1.7 1.07± 0.12
2 35.1 ± 3.0 85.4 ± 7.5 34.8 ± 3.6 2,014 ± 19b 24.7 ± 2.8 38.2 ± 4.2 32.1 ± 2.2 1.02 ± 0.13
5 34.5 ± 2.3 83.9 ± 7.2 36.0 ± 3.5 5,006 ± 16a 23.8 ± 2.5 37.6 ± 3.9 31.9 ± 2.1 1.09 ± 0.14
P-value 0.973 0.986 0.995 <0.001 0.961 0.955 0.980 0.992
Glu 0 34.2 ± 2.2 84.2 ± 6.9d 35.6 ± 3.4 11.0 ± 0.98 24.0 ± 1.9 37.1 ± 3.6 31.6 ± 1.8 1.04 ± 0.13
0.5 35.6 ± 2.4 579 ± 16c 34.9 ± 3.6 11.7 ± 1.3 23.6 ± 2.0 37.4 ± 3.7 31.8 ± 2.1 0.99 ± 0.15
2 33.9 ± 2.4 2,066 ± 23b 36.4 ± 3.8 11.3 ± 1.2 24.5 ± 2.2 38.0 ± 4.0 31.2 ± 1.9 1.07 ± 0.13
5 34.7 ± 2.6 5,063 ± 37a 35.2 ± 3.6 11.5 ± 1.2 24.9 ± 2.3 37.6 ± 3.5 31.3 ± 2.0 1.10 ± 0.14
P-value <0.001 <0.001 0.963 0.994 0.992 0.974 0.988
Arg 0 34.2 ± 2.2b 84.2 ± 6.9 35.6 ± 3.4 11.0 ± 0.98d 24.0 ± 1.9d 37.1 ± 3.6d 31.6 ± 1.8 1.04 ± 0.13d
0.5 34.6 ± 2.3b 83.0 ± 6.6 35.2 ± 3.3 42.4 ± 3.7c 378 ± 16c 69.4 ± 4.2c 32.0 ± 2.0 1.31 ± 0.16c
2 35.8 ± 2.5ab 83.6 ± 7.4 36.7 ± 3.5 76.5 ± 4.9b 1,601 ± 25b 113 ± 7.5b 31.8 ± 2.1 1.84± 0.21b
5 36.5 ± 2.7a 87.5 ± 6.9 36.4 ± 3.8 109 ± 7.4a 3,728 ± 31a 142 ± 8.7a 32.2 ± 2.1 3.60 ± 0.39a
P-value <0.001 0.562 0.954 <0.001 <0.001 <0.001 0.966 <0.001
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1Rumnal fluid was incubated for 2 h in the presence of an added amino acid. These sets of tubes were run simultaneously with those described in Table 1. Values, expressed as nmol/mL of ruminal fluid for AA and as µmol/mL of ruminal fluid for ammonia, are means ± SEM, n = 6. To facilitate comparisons, data for all the “0 mM” groups were taken from the “None addition–2-h incubation” group in Table 1, whereas data for the “5 mM” group of each added AA were taken from the corresponding “5 mM addition–2-h incubation” group in Table 1.

2NH3 plus NH4+.

a–dWithin a column, means not sharing the same superscript differ (P < 0.05), as analyzed by one-way ANOVA, followed by the Student–Newman–Keuls multiple comparison test.

The rates of formation of 14CO2, 14C-Gln, 14C-Asp, 14C-Ala, 14C-ornithine, 14C-Pro, and 14C-protein from extracellular 14C-Glu (5 mM) were negligible and there was no detectable production of 14C-Cit or 14C-Arg from extracellular 14C-Glu, compared with the formation of 14C-labeled products from extracellular 14C-Gln or 14C-Arg (Table 4). There was no formation of 14C-labeled AA (including Asn, Ser, Gly, His, Lys, Thr, taurine, Phe, Leu, Ile, Val, Trp, Tyr, Met, or Cys) from 5 mM 14C-Glu during a 2-h period of incubation. There was no detectable formation of 14CO2 or any 14C-labeled AA (including Glu, Asp, Asn, Ser, Gln, His, Gly, Arg, Thr, taurine, Ala, Phe, Leu, Ile, Val, Trp, Tyr, ornithine, Lys, Pro, Met, or Cys) from extracellular 14C-Cit (5 mM) during a 2-h period of incubation. At the end of the 2-h period of incubation, no urea or 14C-urea was detected in the medium.

Table 4.

Metabolism of 14C-labeled glutamate, citrulline, arginine, and glutamine in incubated ovine ruminal fluid1

Variable l-[U-14C]Glu, 5 mM l-[U-14C]Cit, 5 mM l-[U-14C]Arg, 5 mM l-[U-14C]Gln, 5 mM
Uptake of AA 13.1 ± 1.2c ND3 1,192 ± 88b 1,870 ± 125a
Production of metabolites
 14CO2 2.04 ± 0.18c ND 248 ± 20b 376 ± 24a
 14C-Gln 0.82 ± 0.010b ND 1.75 ± 0.19a —
 14C-Asp 2.55 ± 0.18c ND 27.8 ± 1.6b 39.4 ± 3.1a
 14C-Ala 2.89 ± 0.33b ND 3.44 ± 0.28b 28.5 ± 2.6a
 14C-Glu — ND 4.52 ± 0.41b 1,558 ± 103a
 14C-Pro 0.19 ± 0.015c ND 820 ±75a 4.31 ± 0.35b
 14C-Orn 0.25 ± 0.021c ND 237 ± 18a 16.2 ± 1.8b
 14C-Cit ND ND 96 ± 7.7a 2.66 ± 0.28b
 14C-Arg ND ND — 4.07 ± 0.36a
 14C-Protein2 7.82 ± 0.81c ND 126 ± 6.4b 159 ± 9.1a
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1Ruminal fluid (containing microorganisms) was incubated for 2 h in the presence of a 14C-labeled AA plus an unlabeled AA (5 mM). The specific radioactivity of a labeled AA was 100 dpm/nmol AA. Production of 14CO2 (nmol) was calculated as dpm in 14CO2/the specific radioactivity of a labeled AA expressed as dpm/nmol C. Production of 14C-AA (nmol) was calculated as dpm in 14C-AA/the specific radioactivity of a labeled AA expressed as dpm/nmol AA. Values, expressed as nmol/mL of ruminal fluid per 2 h, are means ± SEM, n = 6. 14C-Ser was produced from 5 mM [U-14C]Gln at the rate of 2.93 ± 0.21 nmol/mL of ruminal fluid per 2 h, but there was no detectable formation of 14C-Ser from the other 14C-labeled AA.

2Incorporation of the labeled AA into protein.

3ND, not detected.

a–cWithin a row, means not sharing the same superscript are different (P < 0.05), as analyzed by one-way ANOVA, followed by the Student–Newman–Keuls multiple comparison test.

In vivo experiment

Concentrations of AA in the plasma of sheep after oral administration of Cit, Gln, or urea are summarized in Tables 5, 6, and 7, respectively. Oral intake of Cit, Gln, or urea (8 g each) did not affect the concentrations of ammonia, urea, glucose, or any AA in plasma other than the Cit and Gln dietary supplement. As baseline values, Gly was consistently the most abundant AA in plasma among all groups of sheep, followed by Gln, Arg, Ala, Pro, Cit, Val, total Cys, and Leu in order of decreasing abundance. Concentrations of Cit in plasma increased progressively by 117% at 4 h, compared with the baseline value (P < 0.001). The increases in concentrations of Arg in plasma of sheep were significant (P < 0.05) at 2 h (+14%) and 4 h (+23%) after oral administration of Cit. Oral administration of Cit did not affect (P > 0.05) the concentrations of other AA in plasma. Oral administration of Gln only slightly (+5.8%) increased (P < 0.05) the concentration of Gln in plasma of sheep at 4 h by 6%, compared with the baseline value.

Table 5.

Concentrations of AA, ammonia, urea, and glucose in plasma of sheep after consumption of a l-Cit supplement1

Time after Cit administration
AA 0 h 0.5 h 1 h 2 h 4 h P-value
Asp 11 ± 1.1 11 ± 0.7 10 ± 0.8 10 ± 1.1 11 ± 1.2 0.973
Glu 61 ± 1.2 62 ± 1.9 62 ± 3.5 61 ± 2.4 62 ± 3.0 0.844
Asn 33 ± 3.4 35 ± 3.3 31 ± 2.3 33 ± 3.0 35 ± 3.7 0.809
Ser 75 ± 3.7 79 ± 2.4 74 ± 1.6 77 ± 3.2 79 ± 6.6 0.534
Gln 372 ± 33 385 ± 29 392 ± 32 389 ± 32 386 ± 30 0.784
His 62 ± 7.0 63 ± 7.7 61 ± 8.1 61 ± 7.7 63 ± 8.9 0.932
Gly 511 ± 73 531 ± 79 532 ± 77 515 ± 79 527 ± 74 0.894
Thr 60 ± 5.7 61 ± 6.1 60 ± 3.6 62 ± 5.0 63 ± 4.5 0.715
Cit 140 ± 11d 147 ± 12cd 162 ± 12c 213 ± 16b 304 ± 29a <0.01
Arg 190 ± 25d 195 ± 27cd 202 ± 28c 217 ± 31b 233 ± 32a <0.01
β-Ala 18 ± 3.2 18 ± 3.3 20 ± 2.8 19 ± 3.7 19 ± 3.1 0.902
Taurine 77 ± 4.9 73 ± 5.9 71 ± 5.9 67 ± 4.7 66 ± 6.2 0.702
Ala 182 ± 21 184 ± 26 176 ± 26 173 ± 24 167 ± 22 0.352
Tyr 61 ± 7.8 64 ± 9.0 61 ± 8.1 61 ± 8.2 61 ± 6.9 0.711
Trp 39 ± 1.9 39 ± 2.4 38 ± 2.2 39 ± 2.9 37 ± 1.9 0.569
Met 24 ± 1.6 26 ± 1.5 25 ± 1.7 24 ± 1.8 25 ± 2.1 0.818
Val 128 ± 9.4 137 ± 13 135 ± 10 139 ± 13 139 ± 7.6 0.477
Phe 36 ± 2.4 37 ± 2.7 37 ± 1.9 37 ± 1.9 36 ± 3.7 0.616
Ile 62 ± 3.6 70 ± 3.6 66 ± 5.0 68 ± 2.3 69 ± 2.0 0.343
Leu 107 ± 7.5 115 ± 8.7 113 ± 7.5 112 ± 7.9 108 ± 5.7 0.496
Orn 78 ± 6.6 80 ± 6.9 84 ± 7.0 97 ± 8.3 107 ± 8.6 0.114
Lys 94 ± 13 97 ± 15 94 ± 13 94 ± 13 93 ± 13 0.819
Pro 156 ± 11 154 ± 13 159 ± 14 157 ± 14 152 ± 16 0.996
Total Cys2 114 ± 9.2 118 ± 12 121 ± 10 109 ± 13 116 ± 14 0.963
Ammonia3 91 ± 4.3 93 ± 5.0 88 ± 5.6 95 ± 6.2 90 ± 5.7 0.908
Urea 6.82 ± 0.51 6.94 ± 0.48 7.03 ± 0.64 6.98 ± 0.57 6.63 ± 0.72 0.976
Glucose 3.38 ± 0.28 3.41 ± 0.32 3.45 ± 0.34 3.29 ± 0.30 3.26 ± 0.35 0.981
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1Values, expressed as nmol/mL for AA and ammonia, and as µmol/mL for urea and glucose, are means ± SEM, n = 6. The P-value refers to the ANOVA F-value.

2Cysteine + ½ cystine.

3NH3 plus NH4+.

a–bWithin a row, means not sharing the same superscript differ (P < 0.05), as analyzed by one-way ANOVA for repeated measure data, followed by the Student–Newman–Keuls multiple comparison test.

Table 6.

Concentrations of AA, ammonia, urea, and glucose in plasma of sheep after consumption of a glutamine supplement1

Time after glutamine administration
AA 0 h 0.5 h 1 h 2 h 4 h P-value
Asp 13 ± 1.7 13 ± 1.4 13 ± 1.8 13 ± 1.2 9.8 ± 0.3 0.141
Glu 60 ± 5.8 62 ± 4.3 60 ± 5.4 59 ± 4.5 58 ± 4.7 0.366
Asn 24 ± 2.9 24 ± 2.1 26 ± 2.5 25 ± 2.2 28 ± 2.3 0.285
Ser 72 ± 3.1 77 ± 3.8 77 ± 5.1 78 ± 3.6 76 ± 4.2 0.148
Gln 362 ± 6.2b 364 ± 8.7ab 370 ± 9.0ab 373 ± 8.7ab 383 ± 11a 0.012
His 61 ± 3.9 62 ± 4.1 62 ± 3.6 62 ± 3.7 60 ± 2.7 0.849
Gly 515 ± 82 527 ± 86 528 ± 93 540 ± 98 533 ± 94 0.646
Thr 62 ± 3.8 62 ± 3.3 63 ± 4.3 64 ± 3.7 62 ± 2.7 0.765
Cit 143 ± 14 148 ± 17 142 ± 12 144 ± 14 153 ± 12 0.352
Arg 186 ± 13 193 ± 15 192 ± 14 193 ± 11 196 ± 11 0.383
β-Ala 18 ± 1.8 20 ± 1.3 19 ± 2.3 19 ± 1.5 19 ± 1.3 0.619
Tau 69 ± 5.6 74 ± 6.1 73 ± 4.7 71 ± 3.2 71 ± 4.4 0.988
Ala 186 ± 19 188 ± 18 186 ± 18 178 ± 16 174 ± 15 0.448
Tyr 64 ± 6.5 66 ± 6.9 67 ± 8.4 63 ± 7.2 63 ± 7.3 0.603
Trp 37 ± 2.2 38 ± 2.7 39 ± 2.3 39 ± 2.4 38 ± 2.0 0.814
Met 25 ± 1.8 26 ± 1.5 25 ± 2.1 24 ± 2.1 24 ± 1.3 0.705
Val 125 ± 8.5 133 ± 9.0 136 ± 9.5 133 ± 13 135 ± 14 0.950
Phe 37 ± 2.2 38 ± 1.9 39 ± 1.3 39 ± 3.0 38 ± 2.7 0.677
Ile 67 ± 4.0 70 ± 2.5 70 ± 2.4 70 ± 1.6 69 ± 2.6 0.857
Leu 106 ± 5.8 113 ± 5.2 114 ± 4.7 109 ± 3.9 106 ± 5.7 0.216
Orn 78 ± 2.6 80 ± 2.2 84 ± 3.6 90 ± 2.7 87 ± 2.6 0.437
Lys 93 ± 10 92 ± 9.7 91 ± 9.6 92 ± 9.5 92 ± 11 0.925
Pro 152 ± 14 149 ± 11 155 ± 13 157 ± 14 150 ± 13 0.987
Total Cys2 108 ± 8.5 113 ± 9.7 114 ± 8.8 117 ± 9.3 102 ± 8.0 0.778
Ammonia3 88 ± 5.0 90 ± 5.6 91 ± 5.9 93 ± 6.5 86 ± 6.4 0.930
Urea 6.67 ± 0.58 6.75 ± 0.62 6.81 ± 0.60 6.52 ± 0.73 6.44 ± 0.69 0.964
Glucose 3.41 ± 0.26 3.37 ± 0.23 3.48 ± 0.27 3.53 ± 0.28 3.32 ± 0.25 0.985
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1Values, expressed as nmol/mL for AA and ammonia, and as µmol/mL for urea and glucose, are means ± SEM, n = 6. The P-value refers to the ANOVA F-value.

2Cysteine + ½ cystine.

3NH3 plus NH4+.

a–bWithin a row, means not sharing the same superscript differ (P < 0.05), as analyzed by one-way ANOVA for repeated measure data, followed by the Student–Newman–Keuls multiple comparison test.

Table 7.

Concentrations of AA, ammonia, urea, and glucose in plasma of sheep after consumption of a urea supplement1

Time after urea administration
AA 0 h 0.5 h 1 h 2 h 4 h P-value
Asp 11 ± 0.8 11 ± 0.7 9.9 ± 1.0 10.6 ± 0.7 10 ± 0.6 0.609
Glu 59 ± 4.0 55 ± 3.9 51 ± 3.3 55 ± 2.9 52 ± 3.0 0.405
Asn 29 ± 2.7 27 ± 2.1 26 ± 2.5 26 ± 2.3 30 ± 2.2 0.689
Ser 76 ± 2.7 75 ± 2.9 74 ± 4.1 75 ± 3.7 76 ± 4.8 0.826
Gln 377 ± 41 382 ± 41 385 ± 42 388 ± 43 387 ± 44 0.621
His 61 ± 3.2 60 ± 2.4 60 ± 2.5 60 ± 2.7 59 ± 3.7 0.861
Gly 517 ± 77 512 ± 68 526 ± 68 531 ± 76 542 ± 76 0.845
Thr 60 ± 3.0 61 ± 2.4 62 ± 2.5 61 ± 3.7 60 ± 3.8 0.596
Cit 143 ± 11 140 ± 8.7 142 ± 9.1 142 ± 9.2 149 ± 9.9 0.701
Arg 188 ± 23 189 ± 23 189 ± 21 184 ± 25 193 ± 23 0.824
β-Ala 18 ± 0.6 19 ± 1.3 18 ± 1.5 19 ± 1.5 18 ± 0.7 0.981
Tau 73 ± 7.1 73 ± 8.2 72 ± 8.2 73 ± 6.1 70 ± 7.4 0.463
Ala 180 ± 26 181 ± 26 179 ± 27 168 ± 27 162 ± 25 0.301
Tyr 60 ± 4.0 61 ± 4.4 60 ± 4.7 59 ± 4.2 59 ± 4.6 0.327
Trp 38 ± 3.2 39 ± 3.2 38 ± 3.5 37 ± 2.5 38 ± 3.0 0.924
Met 23 ± 1.6 23 ± 1.3 23 ± 1.5 22 ± 1.8 23 ± 1.7 0.716
Val 129 ± 12 132 ± 10 129 ± 9.2 123 ± 4.8 121 ± 5.0 0.945
Phe 38 ± 1.3 39 ± 1.8 39 ± 2.2 38 ± 2.5 39 ± 2.6 0.226
Ile 68 ± 4.2 69 ± 3.6 67 ± 2.9 66 ± 3.0 64 ± 3.1 0.618
Leu 107 ± 3.5 112 ± 5.5 108 ± 4.6 104 ± 3.3 102 ± 3.4 0.224
Orn 79 ± 6.8 80 ± 8.0 79 ± 4.6 78 ± 3.0 80 ± 2.7 0.852
Lys 93 ± 6.7 95 ± 7.0 96 ± 7.3 94 ± 6.0 91 ± 5.5 0.538
Pro 144 ± 10 147 ± 11 148 ± 9.7 151 ± 12 142 ± 9.2 0.973
Total Cys2 110 ± 8.6 112 ± 9.0 114 ± 10 119 ± 11 106 ± 9.8 0.910
Ammonia3 90 ± 5.1 91 ± 5.8 96 ± 6.3 98 ± 6.5 93 ± 6.0 0.744
Urea 6.63 ± 0.49 6.71 ± 0.55 6.88 ± 0.57 6.92 ± 0.64 6.57 ± 0.52 0.978
Glucose 3.46 ± 0.25 3.40 ± 0.27 3.51 ± 0.26 3.65 ± 0.29 3.40 ± 0.28 0.964
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1Values, expressed as nmol/mL for AA and ammonia, and as µmol/mL for urea and glucose, are means ± SEM, n = 6. Data were analyzed by one-way ANOVA for repeated measure data, followed by the Student–Newman–Keuls multiple comparison test. The P-value refers to the ANOVA F-value.

2Cysteine + ½ cystine.

3NH3 plus NH4+.

Discussion

Both Cit and Arg are intermediates of the hepatic urea cycle in mammals (including cattle). Although Arg is formed from Cit via this metabolic pathway, there is no net synthesis of Arg by the liver under physiological conditions because Arg is rapidly hydrolyzed by hepatic arginase into ornithine and urea (Wu and Morris, 1998). There is evidence that the Arg pool in the urea cycle is not mixed with the extracellular Arg pool due to complex metabolic channeling in the liver (Srere, 1987). At present, it is not clear whether Arg of the urea cycle pool is used for syntheses of protein, creatine, nitric oxide, and polyamines. In adult animals, net synthesis of Arg from Cit occurs primarily in the kidneys and, to a lesser extent, in other extrahepatic tissues and cells (e.g., endothelial cells and macrophages) to provide Arg for metabolic utilization (Wu and Morris, 1998).

Ruminal bacteria extensively catabolize Arg (Owens and Bergen, 1983). Thus, dietary supplementation with rumen-unprotected Arg is not effective in increasing the concentration of Arg in the plasma of ruminants, including sheep. As with nonruminant animals (Wu et al., 2018), results of recent studies indicate that augmenting the provision of Arg to sheep either through intravenous administration (Lassala et al., 2010, 2011; Satterfield et al., 2012, 2013; McCoard et al., 2014; Sales et al., 2016) or in the rumen-protected form (Hassan et al., 2011; Al-Rubeii et al., 2015; de Chávez et al., 2015; Zhang et al., 2016a; Ashour et al., 2018; Peine et al., 2018) can improve growth, fertility, and lactation performance of sheep. Arginine acts via activating the mechanistic target of rapamycin cell signaling pathway and promoting polyamine-dependent protein synthesis in skeletal muscle (Yao et al., 2008), placenta (Kim et al., 2011a, 2011b; Wang et al., 2014, 2015a, 2015b, 2016), mammary epithelial cells (Ma et al., 2018), and development of brown adipocytes in fetuses (Ma et al., 2017). Thus, there is increasing interest in the use of rumen-protected Arg as a dietary supplement to improve health, growth, production efficiencies, and meat quality of sheep (Prezotto et al., 2018; Zhang et al., 2018, 2019a; Reynolds et al., 2019) and other ruminants (Green et al., 2017; Meyer et al., 2018; Teixeira et al., 2019). A novel and important finding from the present study is that Cit, which is converted into Arg in mammals at a nearly 100% efficiency (Wu and Morris, 1998; Lassala et al., 2009), is not degraded by ruminal microbes of sheep, like we reported for cattle (Gilbreath et al., 2019, 2020). The metabolic patterns of extracellular AA in the ruminal fluid were generally similar between adult sheep fed the soybean hulls-, wheat middlings-, and corn-based diet (Satterfield et al., 2013) and hay-fed adult steers (Gilbreath et al., 2019), except that the rates of Gln and Arg utilization by ruminal microbes were 23% and 73% greater, respectively, in the sheep than those in the cattle. This work advances our knowledge of AA metabolism in ruminants and provides a practical new means to feed sheep with unencapsulated Cit for enhancing the Arg availability for metabolism in their tissues.

Results with 14C-Glu studies revealed negligible degradation of Glu by the mixed ovine ruminal microbes (Table 4), like we reported for bovine ruminal microbes (Gilbreath et al., 2019). The ruminal bacterium Streptococcus bovis may contribute to a slow rate of utilization of extracellular Glu (Hen and Russell, 1989). While the ruminal microbes of steers metabolized little or no extracellular Glu, Gln-derived intracellular Glu can be degraded to aspartate and ornithine (Table 2). However, most of the Gln-derived Glu accumulated in the rumen fluid, possibly due to a limited availability of intracellular α-ketoacids for initiating Glu transamination in the microbes. As reported for bovine ruminal microbes (Gilbreath et al., 2019), ovine ruminal microbes do not take up extracellular Cit (Table 4), possibly due to the lack of a transporter. In support of this view, Stalon and Merceniner (1984) found that few bacteria can utilize extracellular Cit as a nitrogen source for growth. Of note, the mammalian liver does not take up extracellular Cit (Wu and Morris, 1998) and red blood cells of adult rats and humans do not take up extracellular Glu (Watford, 2002).

To our knowledge, this is the first report on the degradability of extracellular Cit and Glu by mixed microbes in the rumen of sheep. Chalupa (1976) demonstrated that bovine rumen fluid, when incubated in the presence of 2% starch plus 8 mM urea, utilized extracellular Glu based on the disappearance of Glu from the medium over a 6-h period. Those results were cautiously interpreted by the author as the “apparent degradation” of Glu by microbes in the rumen, due to the lack of evidence for true degradation of this AA such as the formation of metabolites from Glu. Chalupa (1976) provided urea (a ready precursor of ammonia) and starch (a ready source of carbon skeletons including α-ketoacids) to support synthetic processes; therefore, the disappearance of Glu from the incubation medium may have resulted primarily from the utilization of extracellular Glu for Gln, production by Gln synthetase in ruminal fluid, as well as protein and peptide synthesis by microbes in the rumen, rather than degradation of extracellular Glu. In sheep, orally administered Glu and Cit may effectively enter the small intestine to exert their functions on the mucosa, as demonstrated in nonruminants (Wu, 2013; Hou and Wu, 2018). In support of this, dietary supplementation with ~1% Cit to adult sheep increased the concentrations of Cit and Arg in plasma by 117% and 23%, respectively, at 4 h after consuming the feed (Table 5). Similarly, dietary supplementation with 0.25% Cit to adult steers enhanced the concentrations of Cit and Arg in plasma by 22% and 14%, respectively, at 4 h after consuming the feed (Gilbreath et al., 2020). Therefore, there is no need to encapsulate Cit or Glu in a rumen-protected form when the AA are supplemented to the diet of ruminants. Dietary Cit, an effective precursor of arginine in ruminants, elicits physiological responses such as improvements in embryonic survival, immunity, and lactation, as reported for swine (Wu et al., 2018). With such a simple means to provide ruminants with dietary Cit and Glu, there is no need to manufacture rumen-protected arginine using challenging and expensive technologies.

Oral administration of Gln (~1% of the diet) slightly enhanced the concentration of Gln in plasma of sheep at 4 h (Table 5). This may not be interpreted to indicate that Gln escapes the rumen to enter the blood. It is possible that orally administered Gln was utilized by bacteria in the rumen to synthesize AA (including Glu, Ala, Asp, and branched-chain AA), which subsequently served as substrates for protein synthesis. The microbial protein would then be terminally hydrolyzed by proteinases and peptidases in the small intestine to release Gln and other AA (Butter and Folds, 1985; Owens and Bergen, 1983). We reported that dietary supplementation with 0.25% Gln (in an unprotected form) to adult steers did not affect the concentrations of Gln in plasma (Gilbreath et al., 2020). Of note, oral intake of 8 g urea was not effective in enhancing the concentrations of any AA in the plasma of sheep within a 4-h period likely due to no change in the ruminal synthesis of microbial proteins. Because only a small amount of Cit, Gln, or urea (8 g) was fed to sheep, each supplement did not affect the concentrations of ammonia, urea, or glucose in plasma (Tables 5–7).

In summary, the results of our in vitro and in vivo experiments indicated that, in contrast to Gln and Arg, extracellular Cit was not degraded by ovine ruminal microbes due to the lack of uptake and that extracellular Glu underwent little catabolism by the cells due to limited uptake. Oral administration of Cit effectively increased the concentrations of both Cit and Arg in the plasma of adult sheep, as we reported previously for nonruminant animals and adult cattle. This new knowledge is expected to advance the field of AA nutrition in ruminants and aid in the development of dietary supplementation with Cit or Glu to possibly enhance their growth and productivity.

Acknowledgments

This work was supported by the Texas A&M AgriLife Research Beef Program and the Department of Animal Science Mini-Grant Program. We thank Gayan I. Nawaratna, W. David Long, Neil D. Wu, and Shengdi Hu for technical assistance.

Glossary

Abbreviations

AA

amino acids

Cit

l-citrulline

HPLC

high-performance liquid chromatography

NDF

neutral detergent fiber

Conflict of interest statement

The authors declared no conflict of interest.

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