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. 2022 Mar 11;100(4):skac077. doi: 10.1093/jas/skac077

Equine enterocytes actively oxidize l-glutamine, but do not synthesize l-citrulline or l-arginine from l-glutamine or l-proline in vitro

Rafael E Martinez 1,✉, Jessica L Leatherwood 1,2, Amanda N Bradbery 3, Brittany L Silvers 1, Jennifer Fridley 2, Carolyn E Arnold 2, Erin A Posey 1, Wenliang He 1, Fuller W Bazer 1, Guoyao Wu 1
PMCID: PMC9030134  PMID: 35275603

Abstract

In livestock species, the enterocytes of the small intestine are responsible for the synthesis of citrulline and arginine from glutamine and proline. At present, little is known about de novo synthesis of citrulline and arginine in horses. To test the hypothesis that horses of different age groups can utilize glutamine and proline for the de novo synthesis of citrulline and arginine, jejunal enterocytes from 19 horses of three different age groups: neonates (n = 4; 7.54 ± 2.36 d of age), adults (n = 9; 6.4 ± 0.35 yr), and aged (n = 6; 22.9 ± 1.0 yr) with healthy gastrointestinal tracts were used in the present study. Enterocytes were isolated from the jejunum and incubated at 37 °C for 30 min in oxygenated (95% O2/5% CO2) Krebs bicarbonate buffer (pH 7.4) containing 5 mM D-glucose and 0 mM, 2-mM L-[U-14C]glutamine, or 2 mM L-[U-14C]proline plus 2 mM L-glutamine. Concentrations of arginine, citrulline, and ornithine in cells plus medium were determined using high-performance liquid chromatography. Results indicate that the rate of oxidation of glutamine to CO2 was high in enterocytes from neonatal horses, but low in cells from adult and aged horses. Enterocytes from all age groups of horses did not degrade proline into CO2. Regardless of age, equine enterocytes formed ornithine from glutamine and proline, but failed to convert ornithine into citrulline and arginine. Because arginine is an essential substrate for the synthesis of not only proteins, but also nitrogenous metabolites (e.g., nitric oxide, polyamines, and creatine), our novel findings have important implications for the nutrition, performance, and health of horses.

Keywords: arginine, citrulline, enterocyte, equine, ornithine, small intestine

Lay Summary

The amino acid arginine (Arg) is a precursor for the synthesis of multiple biological molecules including nitric oxide, polyamines, and creatine that are involved in cell proliferation, cellular remodeling, dilation of blood vessels, and phosphocreatine production for a readily available source of energy. Multipurpose capabilities of Arg have increased the interest in its effects in other species and must be evaluated in the horse. Levels of Arg are deficient in the milk of mammals such as humans, cows, sheep, and pigs, but their neonates are capable of synthesizing citrulline and Arg from glutamine and proline in the small intestine. High concentrations of Arg in milk have been observed in the horse, warranting investigation in case that the foal cannot synthesize Arg to support growth and thus rely on milk as the sole source of Arg. To date, no research has determined the endogenous production of Arg in horses to support metabolic and physiological processes; therefore, our experiment quantifies the synthesis of Arg in enterocytes of the small intestine of neonatal, adult, and aged horses. Data collected from this study serve as the necessary first step to determine the Arg requirement in the horse that has over-reaching implications to improve the growth, performance, reproductive efficiency, and to enhance longevity of the horse.

Establishing a specific dietary arginine requirement for the horse has yet to be investigated; therefore, the requirement for supplemental arginine in the equine diet should be determined by experimentally quantifying the endogenous synthesis of this amino acid to address dietary deficiencies.

Introduction

Currently, there is no known dietary requirement for arginine in the horse at any stage of development or physiological state (NRC, 2007), and little is known about its ability to endogenously synthesize arginine de novo. In other mammalian species, such as pigs, humans, rats, cattle, and sheep, arginine is formed from glutamine and proline via the generation of citrulline in the enterocytes of the small intestine (Wu et al., 1994; Wu and Morris, 1998; Bertolo and Burrin, 2008; Gilbreath et al., 2021; Wu, 2022). Accordingly, in contrast to the milk of most mammals that is deficient in arginine (e.g., 40, 36, 34, and 34 mg/g of total amino acids in the milk of sows, humans, cows, and sheep, respectively; Wu and Knabe, 1994; Davis et al., 1994a,b; Wu, 2022), cats have a limited ability to synthesize arginine de novo due to a deficiency of pyrroline-5-carboxylate (P5C) synthase in enterocytes (Rogers and Phang, 1985). However, feline milk contains a high concentration of arginine (64 mg/g of total amino acids, Davis et al., 1994a,b) to compensate for the limited endogenous synthesis of this amino acid (Rogers and Phang, 1985).

In animals, arginine serves not only as a building block for proteins, but also as a precursor for the synthesis of many biologically active molecules including nitric oxide (NO), polyamines, creatine, homoarginine, creatinine, and agmatine (Wu and Morris,1998; Wu et al., 2013; Hou et al., 2016). Additionally, arginine supplementation reduced white fat gain, increased skeletal muscle, decreased serum triglycerides, enhanced insulin sensitivity, and increased brown fat tissue in obese rats (Jobgen et al., 2009), and improved cardiovascular, reproductive, pulmonary, renal, digestive, and immune functions in humans (Wu et al., 2021). Of particular note, arginine prevented fetal growth retardation in rats and underfed ewes and increased birth weights of rats and pigs (Vosatka et al., 1998; Mateo et al., 2007; Gilbreath et al., 2021).

Because of the versatile roles of arginine, there is a growing interest in its nutritional value and metabolism in animals. Increasing evidence shows that dietary supplementation of arginine to animals is not only beneficial for health and productivity under certain physiological conditions such as growth, lactation, and pregnancy (Wu et al., 2009; Wu et al., 2021, 2022). Interestingly, arginine is abundant in the milk of mature mares (60 mg/g of total amino acids) (Davis et al., 1994a), suggesting a possibility that, as for cats, there is little or no synthesis of arginine in horses (Wu, 2022). However, experimental evidence is required to support this proposition. Therefore, we conducted the present study to test the hypothesis that de novo synthesis of citrulline and arginine is limited or absent in enterocytes of young and adult horses.

Materials and Methods

Animal use protocol was not required by the Institutional Animal Care and Use Committee at Texas A&M University as no manipulation of live animals was performed, and animals were not euthanized for the purpose of this study.

Chemicals

l-proline, l-glutamine, o-phthaldialdehyde (OPA), bovine serum albumin (BSA; fraction V, essentially fatty acid free), N-2-hydroxyethylpiperazine N’-2-ethanesulfonic acid (HEPES), and EDTA (disodium) were obtained from Sigma Chemicals (St. Louis, MO). l-[U-14C]glutamine and l-[U-14C]proline were purchased from American Radiolabeled Chemicals (St. Louis, MO). Before use, the radioactive tracers were purified by anion-exchange chromatography as previously described (Wu 1997). High-performance liquid chromatography (HPLC)-grade methanol and water were obtained from Fisher Scientific (Houston, TX).

Collection of samples from the equine jejunum

Jejunum samples were harvested from 19 stock-type horses of three different age groups (Table 1). All horses were euthanized for reasons outside the scope of this current study and for reasons not pertaining to small-intestinal function and health. Tissues were obtained from the necropsy laboratory at the Texas A&M Large Animal Hospital (College Station, TX) and the Veterinary Medical Park at Texas A&M University (College Station, TX).

Table 1.

Age, sex, bodyweight (kg), and body condition score (BCS) of horses

Group Age1 Sex BW1 BCS3
Neonates 7.54 ± 2.36 d 3 colts, 
1 filly ND2 ND2
Adults 6.2 ± 0.67 yr 3 geldings, 6 mares 502.2 ± 22.3 6.0 ± 0.53
Geriatrics 22.9 ± 3.0 yr 1 gelding, 5 mares 424.0 ± 71.5 4.58 ± 0.36
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Values (mean ± SEM).

ND, data not obtained.

Obtained using the 1 to 9 scale (Henneke et al., 1983)

Preparation and incubation of enterocytes from the equine jejunum

In the horse, the jejunum represents the longest portion (~80%) of the small intestine. Therefore, the jejunum was selected for the isolation of enterocytes in this study. Enterocytes were isolated from the equine jejunum as described for the porcine jejunum (Wu et al., 1994). A mid-jejunal segment (50 cm) was obtained from each horse immediately following euthanasia and rinsed thoroughly with 0.9% saline. The jejunum was filled with calcium-free oxygenated (95% O2/5% CO2) Krebs-Henseleit bicarbonate (KHB) buffer (119 mmol/L NaCl, 4.8 mmol/L KCl, 1.2 mmol/L MgSO4, 1.2 mmol/L KH2PO4 and 25 mmol/L NaHCO3, pH 7.4) containing 20 mmol/L HEPES (pH 7.4), 5 mmol/L EDTA (disodium), and 5 mmol/L d-glucose, and was incubated for 30 min at 37 °C in a shaking water bath (70 oscillations/min). Thereafter, the jejunum was gently patted with fingertips for 1 min and its luminal fluid was drained into a 50-mL polystyrene tube. Enterocyte pellets were then obtained by centrifugation (400 × g, 2 min) and washed three times with 20 mL oxygenated KHB buffer (95% O2/5% CO2) containing 2.5 mmol/L CaCl2 (no EDTA), 20 mmol/L HEPES (pH 7.4), and 5 mmol/L d-glucose, and then suspended in fresh KHB buffer. Examination of the isolated equine enterocytes under a dissecting 40× microscope (Fisher Scientific, Houston) showed that they were predominantly (> 90%) enterocytes of columnar shape. The viability of prepared enterocytes was > 95%, as assessed by trypan blue exclusion analyses (Wu et al., 1994; Baldwin and McLeod, 2000).

Incubation of equine enterocytes

Incubations of equine enterocytes were performed in 15-mL polypropylene tubes (round-bottom, 17 × 120 mm; Fisher Scientific, Houston, USA) placed in a shaking water bath (70 oscillations/min). Cells (5 × l06 cells/mL) were incubated at 37 °C for 0 or 30 min in 2 mL of KHB buffer (pH 7.4, saturated with 95%O2/5% CO2, vol/vol) containing 20 mM HEPES, 1% BSA, 5 mM glucose, and either 0 mM (control medium), 2 mM L-[U-14C]glutamine, or 2 mM l-[U-14C]proline plus 2 mM l-glutamine (150 dpm/nmol). Glutamine (2 mM) was added to the 2 mM l-[U-14C]proline medium to provide both ammonia and glutamate to convert the proline-derived P5C possibly into citrulline (Wu, 1997). No tubes with 2 mM l-[U-14C]proline alone were included in the study. The cell incubations were replicated in three tubes within animal. A center-well, which was connected to the rubber stopper of a flask, was suspended within the flask for the collection of 14CO2. At the end of the 30-min incubation, 0.2 mL of Soluene was added through the sealed rubber stopper into the suspended center-well, followed by the addition, through the sealed rubber stopper, of 0.2 mL of 1.5 mol/L HClO4 to the incubation medium. The radioactivity of 14CO2 trapped into Soluene was determined using a liquid scintillation counter (Zhu et al., 2021). We adopted a 30-min period for the incubation of equine enterocytes because the results of our preliminary experiment indicated that the oxidation of 2 mM l-[U-14C]glutamine to 14CO2 by these cells was linear during this time period, as reported for porcine enterocytes (Wu et al., 1995).

Preparation and incubation of enterocytes from the porcine jejunum

To ensure that a lack of synthesis of citrulline and arginine from glutamine and proline in equine enterocytes was not due to an artifact of our cell incubation system, we isolated enterocytes from the jejunum of 7-d-old pigs as described previously (Wu et al., 1994). Incubations of porcine enterocytes in the presence of 2 mM l-[U-14C]glutamine (150 dpm/nmol) and the collection of 14CO2 were the same as described above for equine enterocytes.

Amino acid analysis by HPLC

Acidified medius plus cell content was neutralized with 0.1 mL of 2 mol/L K2CO3 and used for amino acid analysis by HPLC (Wu, 1993; Wu et al., 1994). The HPLC apparatus and precolumn derivatization of amino acids with OPA were as described previously (Wu and Meininger, 2008). Amino acids (except proline and cysteine) were separated on a Supelco 3-µm reversed-phase C18 column (4.6 × 150 mm, I.D.) guarded by a Supelco 40-µm reversed-phase C18 column (4.6 × 50 mm, I.D.). The HPLC mobile phase consisted of solvent A (0.1 mM sodium acetate-0.5% tetrahydrofuran-9% methanol; pH 7.2) and solvent B (methanol), with a combined total flow rate of 1.1 mL/min. A gradient program with a total running time of 49 min (including the time for column regeneration) was developed for satisfactory separation of amino acids (0 min, 14% B; 15 min, 14% B; 20 min, 30% B; 24 min, 35% B; 26 min, 47% B; 34 min, 50% B; 38 min, 70% B; 40 min, 100% B; 42 min, 100% B; 42.1 min, 14% B; 48.5 min, 14% B). Proline was measured by an HPLC method involving oxidation of proline to 4-amino-1-butanol and precolumn derivatization with OPA (Wu, 1993). For cysteine analysis, 100-µL sample was mixed with 50 µL of 50 mM iodoacetic acid (an alkylating agent) for 5 min at room temperature, to convert cysteine to S-carboxymethylcysteine. The latter then reacts with OPA to form a highly fluorescent derivative (Wu et al., 1997). For cystine analysis, 100-µL sample was mixed with 100 µL of 28 mM 2-mercaptoethanol (a reducing agent) for 5 min at room temperature, to convert cystine to cysteine, and the latter was then analyzed as described above. Amino acids were quantified on the basis of authentic standards (Sigma Chemicals, St. Louis, MO) using the Millennium workstation (Waters Inc., Milford, MA). Differences in the concentrations of amino acids in incubation medium plus cell extracts between 0- and 30-min incubation periods in the presence of 2-mM glutamine or 2 mM proline plus 2 mM l-glutamine were used to determine the production of ornithine, citrulline, and arginine by enterocytes.

Statistical analysis

Data were analyzed using the one-way ANOVA in JMP Pro 15 (SAS Inst., Inc., Cary, NC), with the Student–Newman–Keuls multiple comparison test for identifying significant differences among means. For statistical analysis of data from cells without incubation and cells incubated for 0 or 30 min, values were normalized to the baseline (no incubation) before performing one-way analysis of variance, as described previously (Lee et al., 2019). Because data for CO2 production from glutamine were not normally distributed, they were log-transformed for one-way analysis of variance (Assaad et al., 2014). P-values ≤ 0.05 were taken to indicate statistical significance.

Results

Oxidation of glutamine and proline by equine enterocytes

Jejunal enterocytes from horses of all age groups oxidized glutamine to CO2 (Table 2). The rate of glutamine oxidation was high in enterocytes from neonatal horses, but low in cells from adult and aged horses. Interestingly, enterocytes from aged horses oxidized more glutamine to CO2 than cells from adult horses (P < 0.05). There was no production of CO2 from proline by enterocytes from any age group of horses (Table 2).

Table 2.

Production of 14CO2 by equine enterocytes incubated for 30 min with 2 mM l-[U-14C]glutamine or 2 mM l-[U-14C]proline plus 2 mM l-glutamine

Incubation medium Age groups1 P-value
Neonates (n = 4) Adults (n = 9) Aged (n = 6)
2 mM l-[U-14C]Gln 81.1 ± 25.6a 2.47 ± 0.67c 7.07 ± 0.86b 0.01
2 mM l-[U-14C]Pro + Gln ND2 ND ND –
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Values (mean ± SEM) expressed as nmol CO2/106 cells/30 min.

ND, not detected.

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

Synthesis of arginine, citrulline, and ornithine by equine enterocytes in the presence of glutamine or proline

Regardless of age, equine enterocytes synthesized ornithine from glutamine and proline, but failed to convert ornithine into citrulline and arginine (Table 3). The production of ornithine by equine enterocytes incubated in the presence of 2 mM proline plus 2 mM glutamine was greater (P < 0.05) than that in the presence of 2 mM glutamine. The rate of formation of ornithine from glutamine or proline was greater (P < 0.05) in enterocytes from neonatal horses than those from adult or aged horses (Table 3). The rate of formation of ornithine from glutamine or proline was greater (P < 0.05) in enterocytes from aged horses than young horses (Table 3).

Table 3.

Concentrations of arginine, citrulline, and ornithine in incubation medium plus cell extracts of equine enterocytes before and after 30-min incubation period

Age groups1 No Incubation 30-min incubation P-value
0 mM 2 mM Gln 2mM Pro+ 2 mM Gln
Arginine
 Neonates (n = 4) 6.44 ± 0.55b 13.91 ± 2.55a 14.17 ± 3.11a 14.56 ± 3.05a < 0.01
 Adults (n = 9) 7.64 ± 1.42b 13.10 ± 2.50a 12.71 ± 2.35a 13.17 ± 2.56a < 0.01
 Geriatrics (n = 6) 5.51 ± 0.86b 9.78 ± 2.44a 10.23 ± 2.90a 9.91 ± 2.53a < 0.01
Citrulline
 Neonates (n = 4) 0.72 ± 0.19 0.84 ± 0.19 0.76 ± 0.22 0.90 ± 0.13 0.88
 Adults (n = 9) 0.86 ± 0.26 0.92 ± 0.22 0.90 ± 0.19 0.95 ± 0.23 0.85
 Geriatrics (n = 6) 0.67 ± 0.06 0.77 ± 0.12 0.87 ± 0.07 0.81 ± 0.10 0.91
Ornithine
 Neonates (n = 4) 5.24 ± 1.10d 7.75 ± 1.64c 11.84 ± 2.08b 15.1 ± 0.98a < 0.01
 Adults (n = 9) 0.23 ± 0.04d 0.47 ± 0.09c 0.87 ± 0.13b 1.34 ± 0.12a < 0.01
 Geriatrics (n = 6) 1.88 ± 0.62d 2.69 ± 0.93c 4.38 ± 0.91b 6.78 ± 1.65a < 0.01
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Values (mean ± SEM) expressed as nmol/106 cells.

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

Metabolism of glutamine by porcine enterocytes

Jejunal enterocytes from 7-d-old pigs extensively oxidized glutamine to CO2 (Table 4). In contrast to equine enterocytes, porcine enterocytes converted glutamine into ornithine, citrulline, and arginine. The rate of net formation of arginine by porcine enterocytes was greater (P < 0.05) than the rate of net formation of ornithine and citrulline.

Table 4.

Concentrations of arginine, citrulline, and ornithine in incubation medium plus cell extracts of enterocytes from 7-d-old pigs (n = 6) before and after 30-min incubation with 2 mM l-[U-14C]glutamine, and production of 14CO2 from 2 mM l-[U-14C]glutamine

Variable No incubation 30-min incubation
0 mM Gln 2 mM Gln
Amino acid in incubation medium plus cell extracts (mean ± SEM; nmol/106 cells)
Arginine 1.05 ± 0.05c 2.19 ± 0.10b 3.47 ± 0.15a
Citrulline 0.42 ± 0.02c 0.61 ± 0.03b 1.85 ± 0.08a
Ornithine 0.15 ± 0.01c 0.22 ± 0.02b 0.36 ± 0.01a
Production of 14CO2 – – 113.3 ± 6.9
(mean ± SEM; nmol/106 cells/30 min)
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Within a row, means not sharing the same superscript letters are different (P < 0.05), as analyzed by one-way ANOVA for baseline-normalized data, followed by the Student–Newman–Keuls multiple comparison test.

DISCUSSION

Understanding the equine enterocyte’s ability to endogenously produce arginine is of physiological and nutritional importance with respect to the horse’s physiological demands such as athletic performance, reproduction, growth, and longevity. The present study investigated age-related effects on the ability of enterocytes to synthesize arginine, citrulline, and ornithine from glutamine and proline. Similarly, ovine enterocytes are able to oxidize glutamine to CO2 (Oba et al., 2004). Thus, equine enterocytes, as reported for porcine (Wu et al., 1994; Dillon and Wu, 2021) and ovine (Oba et al., 2004) enterocytes, used glutamine as a major metabolic fuel. In contrast, equine enterocytes did not oxidize proline to CO2 possibly due to the absence or limited activity of P5C dehydrogenase in their enterocytes (Wu, 2022).

Enterocytes of the small intestine are exclusively responsible for the synthesis of P5C from glutamine from blood, as well as dietary glutamine and proline in mammals (e.g., pigs, humans, and rats) that are capable of the endogenous synthesis of arginine (Flynn and Wu, 1997; Wakabayashi 1995; Wu 2022). In these cells, P5C undergoes transamination with glutamate to generate ornithine. Enzymes responsible for the conversion of glutamine into ornithine are glutaminase, P5C synthase, and ornithine aminotransferase (OAT), whereas proline is oxidized to ornithine via proline oxidase and OAT. We found that equine enterocytes produced ornithine from glutamine and proline, suggesting that those cells likely express glutaminase, P5C synthase, proline oxidase, and OAT. Results of the current work provide a foundation for future studies, particularly those involving the intravenous administration of 13C- and 15N-labeled glutamine and ornithine into young and adult mares.

In mammalian enterocytes that can produce citrulline from glutamine and proline, ornithine carboxyltransferase (OCT; a mitochondrial enzyme) catalyzes the formation of citrulline from ornithine and carbamoylphosphate. The latter is generated from ammonia and bicarbonate by carbamoylphosphate synthase-I; CPS-I), which is allosterically activated by N-acetylglutamate. Interestingly, enterocytes from young and adult horses failed to convert ornithine into citrulline and arginine. This finding was not due to artifacts in our incubation system for equine enterocytes, because enterocytes from 7-d-old pigs in the same system produced ornithine, citrulline, and arginine from glutamine in the present study, as previously reported for neonatal pigs (Wu et al., 1994). It is possible that equine enterocytes do not express N-acetylglutamate synthase, CPS-I and/or OCT. Thus, there is a species difference in the ability of mammalian enterocytes for endogenous synthesis of citrulline (the immediate precursor of arginine).

All mammalian cells can form arginine from citrulline (Wu and Morris, 1998). The lack of intestinal production of citrulline from glutamine and proline is the metabolic basis for the absence of de novo synthesis of arginine in horses. Thus, the diets of young and adult horses must provide an adequate amount of arginine or citrulline. Because arginine is required for the synthesis of both proteins and many physiologically vital molecules, including polyamines for many cellular functions, creatine (essential for energy metabolism in the skeletal muscle and brain, as well as exercise performance) and nitric oxide (essential for blood flow; Wu 2022), results of the present work have important implications for the nutrition and feeding of horses during their life cycle. Furthermore, our findings explain why the milk of horses, as for the milk of cats, contains unusually large amounts of arginine (Davis et al., 1994a,b) to support the growth, development, and survival of foals during the neonatal period (Figure 1).

Figure 1.

Figure 1.

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Synthesis of l-ornithine from l-glutamine and l-proline in the mitochondria of enterocytes of neonatal, adult, and aged horses. l-Glutamine is hydrolyzed to ammonia plus glutamate. Both l-glutamate and l-proline are converted into ∆1-pyrroline-5-carboxylate, which is subsequently transaminated with l-glutamate to form l-ornithine. The oxidation of glutamine provides both ATP and glutamate for the enzymes catalyzing the formation of l-ornithine, whereas the catabolism of glucose via the pentose cycle supplies NADPH + H+. There is no conversion of l-ornithine into l-citrulline or l-arginine in the equine enterocytes, possibly due to the lack of N-acetylglutamate synthase, carbamoylphosphate synthase-I, and/or ornithine carbamoyltransferase. For comparison, l-glutamine- and l-proline-derived l-ornithine is converted into l-citrulline and l-arginine in enterocytes of young and adult pigs. CP, carbamoyl phosphate; CPS-I, carbamoylphosphate synthase I; CPS-I, carbamoylphosphate synthase I; NAG, l-acetylglutamate; OAA, oxaloacetate; OAT, ornithine aminotransferase; PDG, phosphate-activated glutaminase; P5CS, ∆1-pyrroline-5-carboxylate synthase (a bifunctional enzyme); and SRN, a series of enzyme-catalyzed reactions.

Findings from the current work can guide future in vivo studies of arginine nutrition and metabolism in growing, gestating, lactating, and exercising horses. In the absence of de novo synthesis of arginine, the provision of this amino acid in young and adult horses must depend on their diets or arginine administration. Studies with pigs have shown that dietary supplementation with arginine to neonatal pigs, gestating swine, and lactating sows can enhance their growth rate, embryonic survival, and milk production, respectively (Wu et al., 2009, 2018). Köhne et al. (2018). In addition, Aurich et al. (2019) demonstrated that dietary L-arginine supplementation to mares before and during the initial stages of implantation improved embryonic growth. For example, fetal size from days 25 to 45 after ovulation was greater in mares of reproductive age (young mares: 12.4 ± 0.8 mm and old mares: 14.3 ± 1.4 mm) supplemented with l-arginine compared to unsupplemented control mares (young mares: 10.6 ± 0.6 mm and old mares: 11.4 ± 0.8 mm). In addition, Hunka et al. (2016) reported that dietary supplementation with arginine to lactating mares increased the concentration of glutamine in their milk by ~15%, but these authors did not determine either the milk yield of the mares or the growth performance of foals due to a limited number of animals. At present, we are not aware of any published study concerning the role of dietary arginine supplementation on neonatal growth, milk production, and exercise performance in horses.

In conclusion, the current findings indicated that the enterocytes of horses, regardless of age of horse, did not synthesize citrulline and arginine from glutamine or proline due to the lack of conversion of ornithine into citrulline. As a result of this study, arginine may be considered a nutritionally essential amino acid for horses, especially during physiological states such as performance, pregnancy, and lactation. Future replicated studies are warranted to further investigate the equine enterocyte’s ability to synthesize arginine in vitro and in vivo as well as to determine the expression of all enzymes involved in glutamine and proline catabolism in equine enterocytes.

Acknowledgments

This project was supported by funds provided by the American Quarter Horse Foundation.

Glossary

Abbreviations

BCS

body condition score

BSA

bovine serum albumin

BW

body weight

HEPES

N-2-hydroxyethylpiperazine N’-2-ethanesulfonic acid

HPLC

high-performance liquid chromatography

KHB

Krebs bicarbonate buffer

NO

nitric oxide

OCT

ornithine carboxyltransferase

OPA

o-phthaldialdehyde

P5C

Δ1-pyrroline-5-carboxylate

Conflict of interest statement

The authors affirmatively acknowledge that they were free from influence by any funding sources or their employees that would result in any conflict of interest.

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