Effects of guanidinoacetic acid supplementation on nitrogen retention and methionine flux in cattle

Abstract

Creatine stores high-energy phosphate bonds in muscle and is synthesized in the liver through methylation of guanidinoacetic acid (GAA). Supplementation of GAA may therefore increase methyl group requirements, and this may affect methyl group utilization. Our experiment evaluated the metabolic responses of growing cattle to postruminal supplementation of GAA, in a model where methionine (Met) was deficient, with and without Met supplementation. Seven ruminally cannulated Holstein steers (161 kg initial body weight [BW]) were limit-fed a soybean hull-based diet (2.7 kg/d dry matter) and received continuous abomasal infusions of an essential amino acid (AA) mixture devoid of Met to ensure that no AA besides Met limited animal performance. To provide energy without increasing the microbial protein supply, all steers received ruminal infusions of 200 g/d acetic acid, 200 g/d propionic acid, and 50 g/d butyric acid, as well as abomasal infusions of 300 g/d glucose. Treatments, provided abomasally, were arranged as a 2 × 3 factorial in a split-plot design, and included 0 or 6 g/d of l-Met and 0, 7.5, and 15 g/d of GAA. The experiment included six 10-d periods. Whole body Met flux was measured using continuous jugular infusion of 1-¹³C-l-Met and methyl-²H₃-l-Met. Nitrogen retention was elevated by Met supplementation (P < 0.01). Supplementation with GAA tended to increase N retention when it was supplemented along with Met, but not when it was supplemented without Met. Supplementing GAA linearly increased plasma concentrations of GAA and creatine (P < 0.001), but treatments did not affect urinary excretion of GAA, creatine, or creatinine. Supplementation with Met decreased plasma homocysteine (P < 0.01). Supplementation of GAA tended (P = 0.10) to increase plasma homocysteine when no Met was supplemented, but not when 6 g/d Met was provided. Protein synthesis and protein degradation were both increased by GAA supplementation when no Met was supplemented, but decreased by GAA supplementation when 6 g/d Met were provided. Loss of Met through transsulfuration was increased by Met supplementation, whereas synthesis of Met from remethylation of homocysteine was decreased by Met supplementation. No differences in transmethylation, transsulfuration, or remethylation reactions were observed in response to GAA supplementation. The administration of GAA, when methyl groups are not limiting, has the potential to improve lean tissue deposition and cattle growth.

Introduction

Guanidinoacetic acid (GAA) can be obtained from dietary sources or mainly synthesized endogenously in the kidney from glycine and arginine (Ostojic, 2015). Creatine, because of storing energy in high-energy phosphate bonds, is known for its essential role in cellular energetics (Brosnan et al., 2009). Endogenous synthesis of creatine mainly occurs in the liver and kidney (Wyss and Kaddurah-Daouk, 2000), involving 3 amino acids (AA; arginine, glycine, and methionine [Met]) by a 2-step mechanism (Brosnan and Brosnan, 2004). The first reaction takes place mainly in the kidney, where the transfer of an amidino group from arginine to the amino group of glycine forms ornithine and GAA. GAA then enters into blood to be absorbed by the liver for the subsequent step of creatine synthesis. In the second reaction, a methyl group from S-adenosylmethionine (SAM) in an irreversible reaction is transferred to GAA to form creatine and S-adenosylhomocysteine. Creatine is then released into the blood stream to be taken up by various tissues, but mainly by muscle cells (Wyss and Kaddurah-Daouk, 2000; Riesberg et al., 2016). Also, S-adenosylhomocysteine, resulting from the methylation reaction, is hydrolyzed to form homocysteine. Creatine synthesis is a major consumer of methyl groups in humans (Stead et al., 2006), and GAA supplementation may increase methyl group demand and affect homocysteine metabolism by increasing conversion of SAM to S-adenosylhomocysteine in the body; this can lead to increased plasma homocysteine concentrations in humans (Ostojic, 2014). Some compounds such as Met, choline, and betaine, which play key roles as methyl donors in methylation reactions in the body, may prevent GAA from causing methyl group deficiency and homocysteinemia (Tehlivets et al., 2013).

The production of GAA consumes a large amount of arginine (Brosnan et al., 2011), thus GAA supplementation is able to spare arginine by reducing GAA synthesis. Creatine as a nutritional supplement has the ability to increase muscle mass and BW in humans (Hopwood et al., 2006; Cooper et al., 2012; Gualano et al., 2012), and several studies have illustrated that dietary GAA supplementation can improve growth performance in finishing cattle (Li et al., 2020; Liu et al., 2020, 2021). The body needs creatine for permanent growth of the muscle and for replacing creatine lost by the decomposition of creatine to creatinine, which is excreted in urine (Brosnan et al., 2009; Brosnan and Brosnan, 2010).

Ardalan et al. (2020) showed that GAA supplementation to dairy heifers increased plasma and urinary creatine concentrations and created a mild methyl group deficiency (elevation of plasma homocysteine) that could be prevented with Met supplementation. In the current study, supplementation of GAA was evaluated as a means to improve nitrogen retention (growth) via conversion to creatine, but at the same time it might generate a methyl group deficiency that could have negative consequences. Thus, we evaluated GAA provision under conditions where Met was specifically limiting and also considered how supplemental Met might affect the cattle’s response to GAA.

Our hypotheses were that GAA supplementation to Met-deficient cattle would induce a methyl group deficiency and that Met supplementation would reduce the methyl group deficiency. In light of those expected responses, we further hypothesized that GAA might improve N retention when Met was supplemented.

Materials and Methods

All experimental procedures involving cattle were approved by the Institutional Animal Care and Use Committee at Kansas State University.

Seven ruminally cannulated Holstein steers (161 ± 15 kg initial BW) were used in the experiment. The experiment was designed with 6 steers, and the additional steer was provided a treatment sequence identical to that of another steer. The experiment used a 2 × 3 factorial arrangement of treatments in split-plot design. Methionine supplementation amount (0 or 6 g/d) was the main-plot treatment arranged in 3 concurrent, replicated 2 × 2 Latin squares with 30-d periods; as described below, 0 and 6 g/d of Met were designed to be below and above the Met requirement of the steers. Amount of supplemental GAA (0, 7.5, or 15 g/d) was the subplot treatment, and it was provided in 10-d subplot periods within each main plot period; sequences for the GAA treatment were balanced for carryover among squares. In a previous experiment with growing Holstein heifers (520 kg BW), we observed that doses of GAA up to 40 g/d could be provided without signs of toxicity (Ardalan et al., 2020). The greatest amount of GAA used in this experiment (15 g/d) was similar to that amount on a BW basis.

Prior to the experiment, steers were adapted to the diet for 14 d. After this adaptation, steers were housed in individual metabolism crates that allowed feces and urine to be collected separately. Steers were housed in a temperature-controlled room at 21 °C. All steers had free access to water and were limit-fed twice daily (0700 and 1900 hours).

The experimental approach was based on the Met-deficient model described by Campbell et al. (1997). All steers were maintained under conditions of a Met deficiency through a diet designed to provide deficient amounts of Met (Campbell et al., 1997); the diet principally contained soybean hulls, wheat straw, and molasses (Table 1) and was offered in amounts of 2.7 kg dry matter (DM)/d. The basal diet (Campbell et al., 1997) was formulated to contain only small amounts of ruminally undegraded protein and therefore provided a low metabolizable protein:energy ratio. The diet was designed to provide adequate ruminally degraded protein through ruminal infusion of 10 g urea/d to support maximal microbial growth (Campbell et al., 1997), assuring that changes in nitrogen recycling, which could be affected by treatments, did not affect ruminal microbial growth. To prevent limitations by AA other than Met, all steers received continuous abomasal infusions of a supplemental AA mixture containing deficient amounts of Met. Also, to provide energy and prevent it from being limiting, without increasing microbial protein supply, additional energy was supplied to steers through continuous ruminal infusions of volatile fatty acids and abomasal infusions of 300 g/d glucose. Ruminal infusates for each steer provided 200 g/d acetic acid, 200 g/d propionic acid, and 50 g/d butyric acid as energy sources, and 10 g/d of urea as a source of ruminally available N, with water added to bring the final weight of the mixture to 4 kg/d. The ruminal solution was infused through Tygon tubing (i.d. = 3.32 mm; Saint-Gobain North America, Valley Forge, PA) passed through the ruminal cannula and held in the rumen with a perforated vial attached to the end of the ruminal infusion lines. A peristaltic pump (Model CP-78002-–10; Cole-Parmer Instrument Company, Vernon Hills, IL) was used to make the ruminal and abomasal infusions.

Table 1.

Composition of experimental diet

Ingredient	% of DM
Soybean hulls	82.20
Wheat straw	8.50
Cane molasses	4.18
Premix	5.12
Calcium phosphate	1.96
Sodium bicarbonate	1.25
Calcium carbonate	1.03
Magnesium oxide	0.41
Trace mineral salt1	0.22
Vitamin premix2	0.13
Sulfur	0.10
Selenium premix3	0.010
Bovatec-914	0.018
Nutrient composition
Crude protein	9.3
Organic matter	91.9

¹Composition: >95.5% NaCl, 0.24% Mn, 0.24% Fe, 0.05% Mg, 0.032% Cu, 0.032% Zn, 0.007% I, and 0.004% Co.

²Provided (per kg diet DM): 4,950 IU vitamin A, 3,450 IU vitamin D, and 45 IU vitamin E.

³Provided 0.06 mg Se/kg diet DM.

⁴Supplied 36 mg lasalocid/kg diet DM (Zoetis, Florham Park, NJ).

The basal abomasal infusate containing AA was prepared daily as follows. All steers received a basal infusion of a mixture providing daily 20 g of l-leucine, 15 g of l-isoleucine, 15 g of l-valine, 2 g of l-Met, 20 g of l-lysine-HCl, 15 g of l-threonine, 8 g of l-histidine-HCl-H₂O, 20 g of l-phenylalanine, 5 g of l-tryptophan, 15 g of l-arginine, and 150 g of monosodium glutamate. The branched-chain AA (BCAA: l-valine, l-isoleucine, and l-leucine) was initially solubilized in ~1 kg of water containing 60 g of 6 M HCl. Once BCAA dissolved, the remaining AA, except sodium glutamate, were added and mixed until dissolved. After all AA were dissolved, the sodium glutamate was mixed with the basal AA mixtures and water was added to bring the final weight of the daily infusate to 2 kg.

For preparing the GAA treatments, a 1% (wt/wt) solution of GAA was prepared in water; the GAA was initially solubilized with 0.22 g of 6 M HCl/g GAA, and then 0.11 g of 50% (wt/wt) NaOH/g GAA was added.

All steers received daily supplementation with 10 mg/d folic acid, 0.10 mg/d cyanocobalamin (B₁₂), and 10 mg/d pyridoxine (B₆) to ensure that the steers were not deficient in these vitamins (Lambert et al., 2004). These vitamins were mixed with the abomasal infusate. To prepare the vitamin supplements, 1 g folate was dissolved into 200 mL 50% (wt/wt) acetic acid, and 0.01 g cyanocobalamin and 1 g pyridoxine were dissolved in 200 mL water; 2 mL of each solution were added to each steer’s infusate daily.

Abomasal infusates for each steer were prepared by mixing 2 kg of the basal AA solution, 330 g of dextrose, the amount of GAA solution required to provide the treatment amount of GAA, and 2 mL of each of the 2 vitamin mixtures. Dry l-Met was added to the abomasal infusate based on treatment and allowed to dissolve. Water was then added to bring the total weight of the daily infusate to 4 kg. The abomasal infusate was provided continuously into the abomasum through Tygon tubing (i.d. = 3.32 mm; Saint-Gobain North America) passed through the ruminal cannula, the reticulo-omasal orifice, and the omasum and held in the abomasum with a circular rubber flange (10 cm diameter).

The diet was estimated to provide 2.7 g/d metabolizable Met to the steers, based on measurement of nutrient flows from cattle fed a similar diet (Campbell et al., 1997). Thus, basal supplies of metabolizable Met to all steers were 4.7 g/d (2.7 g/d from the diet plus 2 g/d in the basal abomasal infusions). Two grams per day of Met in the basal infusate were included to prevent the Met deficiency from being so extreme that the steers would be at risk of metabolic derangement. For similar steers, the Met requirement for maximal N retention was 7.9 g/d (Campbell et al., 1997), so the steers receiving no supplemental Met were predicted to be deficient in Met supply, whereas, for steers receiving 6 g/d supplemental Met and having metabolizable Met supplies of 10.7 g/d, the requirement was predicted to be exceeded.

Sample collection and laboratory analyses

Feed analyses

Feed samples were collected from days 4 through 9 of each period and mixed within period to obtain composite samples. Feed samples were dried in a forced-air oven for 72 hr (55 °C) and then ground to pass through a 1-mm screen (Thomas-Wiley Laboratory Mill Model 4, Thomas Scientific, Swedesboro, NJ) and stored for subsequent analysis. Feed refusals, if any, were collected from days 5 through 10 of each period, composited by period, dried at 55 °C in a forced-air oven for 72 hr, and ground to pass a 1-mm screen for later analysis. Total DM of feed and orts were determined by drying samples at 105 °C for 24 hr in a forced-air oven, and ash was determined by combustion at 450 °C for 8 hr.

Blood sampling and analysis

On days 6, 8, and 10 of each period, blood samples were collected from a jugular vein into 10-mL heparinized blood collection tubes (Becton, Dickinson and Co., Franklin Lakes, NJ) at 2 hr after the morning feeding (0900 hours). Samples were stored on crushed ice immediately after collection and then centrifuged (1,000 × g, 4 °C, 15 min) to harvest plasma. Plasma samples were frozen at −20 °C for later analyses of AA, GAA, creatine, and creatinine. Plasma AA (day 10 samples only) were measured by gas chromatography using a commercial kit (EZ:faast; Phenomenex, Torrance, CA) with the exception of homocysteine and cysteine (days 6, 8, and 10 of each period), which were measured by HPLC. Plasma homocysteine and cysteine concentrations were analyzed as 4-fluoro-7-sulfobenzofurazan adducts by reverse-phase HPLC as described by Pfeiffer et al. (1999). Urine and blood GAA, creatine, and creatinine were determined using HPLC according to Shingfield and Offer (1999) with some modification. Plasma samples were prepared by mixing equal volumes of 10% (wt/vol) sulfosalicylic acid and sample, vortexing, freezing overnight, centrifuging (17,000 × g, 10 min, 4 °C), and then filtering through a 0.2-µm syringe filter into HPLC vials for injection. For urine sample preparation, 100 µL of sample was diluted with 900 µL diluent, which consisted of 0.9 g of ammonium phosphate and 1.01 g of sodium 1-heptane sulfonic acid in 1 L of deionized H₂O with pH adjusted to 2.2 with H₃PO₄. Diluted samples were filtered through a 0.2-µm syringe filter into an HPLC vial. The components of the sample were separated on a 25 cm × 4.6 mm Discovery BIO Wide Pore C18 column (5-µm particle size; Supelco, Bellefonte, PA). The mobile phase consisted of 1.01 g sodium 1-heptane sulfonic, 0.9 g ammonium phosphate, 35 mL methanol, and 70 µL triethylamine made to 1 L with deionized H₂O and adjusted to pH 2.8 with 7.5 M H₃PO₄. Compounds were detected by absorbance at 200 nm. To achieve a chromatographic separation, 5 µL sample was injected to the column at 20 °C with a flow rate of 0.5 mL/min for 14 min, then with a flow rate of 1.2 mL/min. Sample separation was completed at 25 min, and the column was flushed with 100% methanol for 10 min at 1.2 mL/min and re-equilibrated with the mobile phase for 19 min. The flow rate was then returned to 0.5 mL/min for 1 min prior to the next injection. Total run time was 55 min.

Nitrogen retention

On days 5 through 10 of each period, total feces and urine for each steer were collected and weighed daily to quantify output. The urinary output was collected into buckets containing 900 mL of 10% (wt/wt) H₂SO₄ to maintain pH below 3 for preventing ammonia loss and limiting microbial growth. Urine and feces were collected, weighed, and sampled daily; 1% of urine and 10% of feces were saved and frozen (–20 °C) for subsequent analysis.

Nitrogen retention was measured over three 2-d collection periods (i.e., days 5 and 6, 7 and 8, and 9 and 10). The total nitrogen content of diet, orts, wet feces, and urine was determined using a combustion analyzer (True Mac; Leco Corporation, St. Joseph, MI). Nitrogen retention was determined as the difference between N intake (feed + infusates − refusals) and N excretion in feces and urine.

Glomerular filtration was calculated with the assumption that creatinine is entirely filtered from the blood and not reabsorbed from glomerulus. Thus, renal reabsorption of GAA was calculated as (1 − ((urinary GAA concentration/urinary creatinine concentration)/(plasma GAA concentration/plasma creatinine concentration))), and renal reabsorption of creatine was calculated similarly with concentrations of creatine replacing those of GAA (Verouti et al., 2021). These renal reabsorptions were calculated for 2-d windows, matching the day 6 plasma concentrations with the urinary concentrations from days 5 and 6, the day 8 plasma concentrations with the urinary concentrations from days 7 and 8, and the day 10 plasma concentrations with the urinary concentrations from days 9 and 10.

Methionine flux measurement

On day 10 of each period, whole body Met flux was measured by continuously intravenously infusing two labeled Met sources (1-¹³C-l-Met [99%] and methyl-²H₃-l-Met [98%], both from Cambridge Isotope Laboratories, Inc., Tewksbury, MA). The labeled Met (0.04 g/hr of each label), following a pulse dose of 0.04 g of each label, were infused through a jugular catheter (MILACATH #LA1420; MILA International, Inc., Florence, KY) that was placed earlier in the day. The infusion period started 4 hr after feeding and lasted 4 hr (Preynat et al., 2009). Blood samples for measuring background enrichment were collected 2 hr prior to initiation of label infusion (i.e., 2 hr after morning feeding) and enriched samples were collected at the end of the 4-hr infusion period into 10-mL heparinized blood collection tubes (Becton, Dickinson and Co., Franklin Lakes, NJ). Samples were stored on crushed ice immediately after collection and then centrifuged (1,000 × g, 4 °C, 15 min) to harvest plasma. Plasma samples were stored at −20 °C for measuring enrichment of label. Enrichments of the 1-¹³C-l-Met and methyl-²H₃-l-Met in plasma were determined using gas-liquid chromatography/mass spectrometry analysis according to Loest et al. (2002).

Methionine flux was calculated according to the procedure of Preynat et al. (2009). The whole body flux of methyl-²H₃-l-Met was defined as: Q(methyl-²H₃-l-Met) = protein synthesis (PS) + transmethylation reactions = protein degradation (PD) + dietary intake + remethylation. The whole body flux rate of 1-¹³C-l-Met was defined as: Q(1-¹³C-l-Met) = PS + transsulfuration = PD + dietary intake. Protein synthesis was calculated as the sum of PD and Met deposition. Methionine deposition was predicted as: N retention, g N/hr × 6.25 g protein/g N × 0.134 mmol Met/g protein (Ainslie et al., 1993). Metabolizable Met intake was calculated as abomasally infused Met plus metabolizable Met provided by the diet (6.71 mmol metabolizable Met/kg DM intake; Campbell et al., 1997).

Statistical analyses

Data were analyzed as a split-plot design using the Mixed procedure of SAS System 9.3 for Windows (SAS Inst. Inc., Cary, NC). For data without repeated measures, fixed effects in the model included main-plot period, Met treatment, sub-plot period within main-plot period, GAA treatment, and Met × GAA. Random effects included steer and steer × main-plot period (as the main-plot error term). For data with repeated measures within a sub-plot period, day and all interactions of day with treatment were included as fixed effects, and day was considered as a repeated measure with the covariance structure of autoregressive (1). Means were separated using polynomial contrasts to test the linear and quadratic effects of GAA as well as the interactions of Met with those effects. Significance was declared at P ≤ 0.05 and tendencies at 0.05 < P ≤ 0.10. Data for 1 steer were removed from periods 2 and 3 due to low feed intake on sampling days. Methionine flux measures were not available for one period due to failure of the pump infusing the label.

Results and Discussion

In our previous research with postruminal supplementation of GAA to cattle (Ardalan et al., 2020), we found evidence of treatment interactions with time for plasma concentration of GAA, creatine, and creatinine, suggesting that cattle had not adapted completely to treatment by day 3 (52 hr after treatment initiation). In the current study with a longer period before the first sampling time, there were no interactions between treatment and time for retention or urinary excretion of nitrogen or for plasma concentrations or renal reabsorptions of GAA, creatine, or creatinine; thus, the presented data for those measures represent all sampling times for those measure (i.e., days 5 through 10 for nitrogen retention and urinary excretion data; days 6, 8, and 10 for plasma concentrations). There was, however, an interaction between treatment and time for plasma homocysteine (data not shown), suggesting that some minor adaptation in plasma metabolites was occurring after day 6. Therefore, only the plasma homocysteine and cysteine data from blood collected on day 10, which would allow the longest adaptation to treatments, are presented.

Dry matter intake, DM and organic matter digestibilities, and N retention

Dry matter intakes were similar among treatments as expected with limit feeding. DM and organic matter digestibilities were increased (P < 0.01) with the administration of Met (Table 2). The greater DM and organic matter digestibilities correspond with less fecal N excretion when Met was supplemented (P < 0.01). In general, we have not observed changes in organic matter digestibility when steers maintained in a similar model (Loest et al., 2002; Awawdeh et al., 2004) were supplemented with Met, and the mechanism whereby Met supplementation increased DM and organic matter digestibilities has no clear explanation. No differences were detected for DM and organic matter digestibilities in response to supplementation of GAA. Opposite of our results, Li et al. (2020), who evaluated effects of increasing levels of GAA (0, 0.3, 0.6, and 0.9 g GAA/kg DM) fed to Angus bulls, indicated that DM intake and ruminal volatile fatty acid concentrations increased when 0.6 or 0.9 g GAA/kg DM was fed. Additionally, feeding 0.9 g GAA/kg DM increased digestibilities of DM and organic matter. The authors suggested that GAA might improve DM intake by increasing nutrient digestibility, ruminal volatile fatty acid concentration, and blood creatine concentration (Li et al., 2020), which can stimulate food intake (Galbraith et al., 2006). Because we infused GAA to the abomasum, it would be unlikely to stimulate ruminal fermentation in our experiment, which could explain our lack of response to GAA for digestibility.

Table 2.

Effect of Met and GAA supplementation on DM intake (DMI), digestibilities, and nitrogen retention

	0 Met			6 g/d Met
	GAA, g/d							P-value 1
Item	0	7.5	15	0	7.5	15	SEM	Met	G-L	G-Q	Met × G-L	Met × G-Q
n	7	7	7	7	6	6
Dietary DMI, kg/d	2.69	2.68	2.66	2.65	2.65	2.66	0.03	0.38	0.87	0.96	0.39	0.71
Digestibility2, %
DM	74.5	74.0	74.2	79.4	77.2	79.6	1.94	<0.01	0.97	0.15	0.76	0.31
Organic matter	77.0	75.9	76.2	80.7	79.0	81.3	1.91	<0.01	0.92	0.15	0.52	0.46
Nitrogen, g/d
Total intake	80.7	83.3	85.7	80.3	83.7	86.8	0.60	0.47	<0.01	0.76	0.19	0.95
Diet	44.4	44.3	44.0	43.8	43.7	44.2	—	—	—	—	—	—
Infused	36.4	39.1	41.7	36.5	40.0	42.6	—	—	—	—	—	—
Urinary	32.3	35.3	37.6	29.3	30.9	31.8	1.62	0.01	<0.01	0.73	0.23	0.98
Fecal	22.0	23.2	22.8	18.2	19.9	18.0	1.59	<0.01	0.64	0.02	0.38	0.35
Retained	26.4	24.9	25.3	32.7	32.8	36.9	2.10	<0.01	0.34	0.29	0.10	0.71

¹G-L, linear effect of GAA; G-Q, quadratic effect of GAA.

²Excludes ruminal and abomasal infusions from calculation of digestibility.

By design, the GAA treatments led to increases in total N intake (Table 2). Urinary (P = 0.01) and fecal (P < 0.01) N excretion decreased when Met was supplemented. The observed reductions in urinary and fecal N resulted in increased (P < 0.01) N retention from 26.4 to 32.7 g/d with Met supplementation, which was expected because Met was designed to be the most limiting AA in our experimental model (Campbell et al., 1997).

A linear (P = 0.001) increase in urinary N and a smaller quadratic (P = 0.02) increase (greatest for the intermediate GAA amount) in fecal N were observed when GAA was supplemented. Nitrogen retention responses to GAA were dependent on the Met status of the steers (Met × GAA-linear, P = 0.10). For steers receiving 6 g/d of Met, there tended to be an increase in N retention in response to increasing GAA supplementation, whereas for steers not receiving supplemental Met there was no response. The lack of a N retention response to 7.5 g/d of GAA in the presence of supplemental Met might have been due to an off-setting decrease in endogenous production of GAA; based on basal urinary excretion of GAA (0.52 g/d), creatine (5.7 g/d), and creatinine (5.7 g/d), endogenous production of GAA should be near 11.5 g/d. Indeed, the 7.5 g/d amount of GAA, because of feedback inhibition of endogenous GAA synthesis (Wyss and Kaddurah-Daouk, 2000), may not have markedly changed total creatine production or methyl group utilization for creatine synthesis. However, at the supplementation level greater than endogenous production (i.e., 15 g/d), the increase in total GAA/creatine availability seemed to improve animal production. In contrast, supplemental GAA had only a nonsignificant, slightly negative effect on nitrogen retention in steers receiving no supplemental Met, perhaps reflecting protein deposition might not be strongly affected by deficiency of methyl-group donors. Taken as a whole, these results support the concept that under conditions of methyl group deficiency, increases in creatine production may not improve nitrogen retention (cattle growth). These observations provide a starting point for further research on the effects of GAA supplementation on increasing lean tissue deposition in growing cattle.

Li et al. (2020) fed Angus bulls a diet based on corn silage and corn grain with dietary GAA at levels of 0, 0.3, 0.6 and 0.9 g/kg DM. Daily gain, BW, and feed conversion improved when GAA was supplemented with the maximal responses to GAA achieved with 0.6 or 0.9 g GAA/kg DM. The steers receiving 0.6 g GAA/kg DM would have consumed about 6 g/d of GAA, with an expectation that only about half of that amount would be available to the steers (Speer et al., 2020); thus, the optimal amount of GAA for steers in the study of Li et al. (2020) was considerably less than in our experiment, and the difference would be even greater if expressed on a BW basis.

In a study conducted by Lemme et al. (2010), broilers received 0.8 g GAA/kg feed or 1.0 g creatine/kg feed in a Met-deficient plant-based diet (i.e., low dietary creatine concentrations) or the same diet supplemented with Met (0.37% vs. 0.57%). For broilers fed Met-adequate diets, GAA and creatine improved weight gain, breast meat yield, and feed intake, whereas GAA and creatine supplementation were ineffective for broilers fed the Met-deficient diet. This demonstrates that GAA and creatine supplements can be more effective when Met is not limiting.

Lemme et al. (2007a) fed male broilers 4 dietary GAA concentrations (0, 0.20, 0.40, or 0.60 g GAA/kg) in a vegetable-based diet. Increasing GAA supplementation increased muscle creatine by about 14%, suggesting that GAA can act as a source of creatine. Additional work by Lemme et al. (2007b) compared the administration of different dietary levels of GAA (0, 0.6, 1.2, 1.8, and 2.4 g GAA/kg) when plant-based diets containing 6% meat and bone meal were fed to broilers. These researchers reported that 0.6 g GAA/kg improved gain:feed and BW gain of female broilers. Also, supplementation with 0.6 g GAA/kg and 1.2 g GAA/kg enhanced breast meat yield in male and female broilers, respectively. These results suggest that dietary supplementation of GAA can improve broiler performance, and the optimal supplementation level of GAA is between 0.6 g GAA/kg and 1.2 g GAA/kg, with an average of 0.7 g GAA/kg diet.

Plasma and urinary concentrations of GAA, creatine, and creatinine

There were no significant interactions between GAA and Met for plasma GAA, creatine, and creatinine. Supplemental GAA linearly (P < 0.01) and quadratically (both 7.5 and 15 g/d GAA led to similar increases; P = 0.02) increased plasma GAA concentrations from 0.45 to 0.62 mg/L (Table 3). These results are in agreement with our previous research conducted on GAA supplementation to dairy heifers (Ardalan et al., 2020). Ardalan et al. (2020) showed that plasma GAA concentrations increased with increasing levels of GAA supplementation from 0.98 to 2.59 mg/L, although the rise in plasma GAA tended to be less when cattle were provided Met. These responses support the conclusion that GAA was absorbed by the gut and made available to the body. These results are also consistent with other reports of GAA administration in humans (Ostojic and Vojvodic-Ostojic, 2015), where GAA administration led to increases in plasma concentrations of GAA.

Table 3.

Effect of Met and GAA supplementation on urinary output and plasma concentrations of GAA, creatine, and creatinine

	0 Met			6 g/d Met
	GAA, g/d							P-value 1
Item	0	7.5	15	0	7.5	15	SEM	Met	G-L	G-Q	Met × G-L	Met × G-Q
n	7	7	7	7	6	6
Urinary, g/d
GAA	0.52	0.74	0.70	0.67	0.62	0.66	0.17	0.97	0.15	0.40	0.14	0.11
Creatine	5.7	7.8	7.1	6.7	6.1	6.5	1.5	0.41	0.33	0.39	0.18	0.07
Creatinine	5.7	7.9	6.9	6.3	6.6	6.9	1.6	0.61	0.13	0.14	0.55	0.13
Plasma, mg/L
GAA	0.45	0.58	0.63	0.45	0.62	0.61	0.046	0.84	<0.01	0.02	0.73	0.43
Creatine	23.8	24.9	26.2	21.1	22.5	24.9	1.1	<0.01	<0.01	0.58	0.20	0.73
Creatinine	6.62	6.94	6.63	6.55	6.52	6.53	0.3	0.32	0.97	0.35	0.94	0.31
Renal reabsorption, %
GAA	−50.1	−28.0	−20.5	−43.9	−12.6	−11.7	19.6	0.64	<0.01	0.22	0.90	0.67
Creatine	71.5	71.0	73.5	66.8	70.3	75.6	2.9	0.61	0.01	0.52	0.11	0.87

¹G-L, linear effect of GAA; G-Q, quadratic effect of GAA.

Plasma creatine concentrations increased linearly (P < 0.01) with increasing levels of GAA supplementation (Table 3). A reduction in plasma creatine concentrations was observed in response to Met supplementation (P < 0.01), suggesting that the greater tissue growth in response to Met might enhance creatine uptake into muscle. Our data are consistent with our previous research (Ardalan et al., 2020) on dairy heifers that demonstrated that plasma creatine concentrations increased at levels up to 30 g/d of supplemental GAA. These data support a conclusion that creatine synthesis was increased in cattle through GAA supplementation. Increases in plasma creatine concentrations in response to GAA supplementation are also consistent with the work of Li et al. (2020), where feeding GAA to bulls increased blood creatine concentrations, suggesting that GAA was utilized for creatine synthesis (Li et al., 2020). There were no treatment effects on plasma creatinine concentrations (P ≥ 0.31), in agreement with research conducted on GAA supplementation to pigs (He et al., 2018) and humans (Ostojic et al., 2013b).

No significant differences among treatments were observed for urinary excretion of GAA, creatine, or creatinine (Table 3). There was, however, a tendency (Met × GAA-quadratic; P = 0.07) for the intermediate level of GAA to increase urinary creatine when no Met was supplemented but to decrease it when 6 g/d Met was provided; a similar pattern was observed for urinary GAA excretion (P = 0.11). Ostojic et al. (2013b) orally supplemented GAA (2.4 g/d) in combination with and without betaine (methyl group donor) to healthy humans and observed increases in serum and urinary creatine when GAA was supplemented with or without supplemental betaine. Supplementation of GAA without betaine increased serum and urinary GAA, but feeding GAA with betaine increased serum GAA but had no effect on urinary excretion of GAA. Thus, when methyl donors are inadequate to support additional methylation of GAA to creatine, GAA is removed via urine. Although statistically weak, our responses for urinary GAA suggest that a similar mechanism may be in place for cattle when Met is provided as the methyl donor.

Both GAA and creatine showed linear increases (Table 3) in renal reabsorption in response to supplemental GAA. However, it is interesting to note that the creatine and GAA reabsorption increased when the plasma concentration of GAA and creatine had also raised following GAA administration. These results are opposite our observations for creatine reabsorption in our previous work (Ardalan et al., 2020), where, in response to GAA supplementation, urine concentration of creatine increased relatively more than did plasma concentration. There are several possible explanations for increasing renal reabsorption of creatine. Both a specific plasma membrane Na⁺/Cl− transporter and a creatine transporter have important roles in tissue uptake and reabsorption of creatine (Jonquel-Chevalier Curt et al., 2015). The creatine transporter is saturable (Walzel et al., 2002), and K_m value for bovine creatine transporter has been determined as 188 µM creatine (25 mg/L; Dodd et al., 1999), although studies evaluating the K_m for creatine transporter have shown varied K_m for different animal species. Therefore, the amount of creatine transporter and the K_m value are important factors in regulating intracellular creatine levels (Dodd et al., 1999). Our plasma creatine concentrations were slightly less than those in our previous work (Ardalan et al., 2020), and near the K_m of the bovine creatine transporter. Therefore, it is possible that some of the discrepancy between the current experiment and previous work is because the greater blood creatine concentrations by Ardalan et al. (2020) led to shifting the clearance of creatine from the muscle to renal excretion.

Creatine and GAA clearance in the body can be affected by others factors such as (1) creatine and GAA competition for reabsorption by the renal tubules (Sims and Seldin, 1949), (2) age (Almeida et al., 2004; Olah et al., 2019), (3) differences among species in glomerular filtration rate (Skotnickaet al., 2007), (4) effect of AA on glomerular filtration rate (Grossman, 1945; Lee and Summerill, 1982), and (5) sex hormones and gender differences (Joncquel-Chevalier Curt et al., 2013). Any of these factors, except species, could have contributed to differences between our previous work and the current experiment in urinary creatine excretion responses as GAA was supplemented.

The body removes excess GAA that does not become methylated to creatine via renal excretion to counteract increases in GAA concentrations (Ostojic et al., 2013b). The very low amounts of GAA in urine demonstrated that most of the supplemental GAA was used for creatine synthesis. Ardalan et al. (2020) observed increases in urinary concentrations of GAA and creatine when up to 30 g/d GAA was supplemented, but the increases in urinary concentrations of GAA were small compared with the increases in urinary creatine. These data demonstrate the effectiveness of GAA for creatine formation.

Tossenberger et al. (2016) observed that broiler chicks fed 0.6 g GAA/kg feed (38 mg/kg BW^0.75 daily) or 6.0 g GAA/kg feed (359 mg/kg^0.75 daily) for 35 d, demonstrated increases in urinary excretion of GAA, creatine, and creatinine. Increases in urinary excretion of GAA, creatine, and creatinine by birds fed 0.6 g GAA/kg feed represented 16.4%, 2.5%, and 3.8%, respectively, of the supplemental GAA, whereas for birds fed 6.0 g GAA/kg feed the urinary losses of GAA, creatine, and creatinine represented 27.6%, 7.1%, and 18.3% of the supplemental GAA. These data demonstrate that renal excretion is a route of elimination of excess GAA and its metabolites creatine and creatinine in chicks.

Methionine flux

The body’s Met pool, which is derived from dietary sources, PD, and de novo synthesis (homocysteine remethylation), can be used for PS or transmethylation reactions. In our experiment, the methyl-labeled Met would be removed from the Met pool through PS or in transmethylation reactions. In contrast, 1-¹³C-l-Met is only removed from the Met pool through PS and transsulfuration reactions, because the 1-¹³C-label is not removed during transmethylation. Subsequent to transmethylation, removal of the 1-¹³C-label does not occur if the labeled homocysteine is remethylated to Met, but the label is lost from the Met pool (contributing to the flux of 1-¹³C-Met) during transsulfuration. This differential partitioning of the 2 labeled Met molecules allows estimation of Met movement through reactions of transmethylation, transsulfuration, and remethylation of homocysteine.

The effects of GAA and Met supplementation on Met flux and related measures are shown in Table 4. Methionine yielded a number of significant main-effect responses, whereas GAA yielded no main-effect responses. There were some significant interactions between the GAA and Met treatments, but the larger magnitude responses to Met are discussed first, followed by a discussion of the interactions between GAA and Met.

Table 4.

Effect of Met and GAA supplementation on measurements derived from Met flux

	0 Met			6 g/d Met
	GAA, g/d							P-value 1
Flux, mmol Met/hr	0	7.5	15	0	7.5	15	SEM	Met	G-L	G-Q	Met × G-L	Met × G-Q
n	6	6	6	6	5	5
1-13C-l-Met	8.02	10.08	9.94	13.71	11.95	11.10	1.18	<0.01	0.73	0.71	0.04	0.38
Methyl-2H3-l-Met	10.68	12.74	13.18	13.67	12.90	12.59	0.86	0.17	0.34	0.65	0.03	0.42
Metabolizable Met2	1.31	1.31	1.30	2.97	2.97	2.99	0.01	<0.001	0.75	0.69	0.21	0.53
Met deposition3	0.95	0.86	0.92	1.14	1.13	1.31	0.09	<0.01	0.34	0.21	0.19	0.88
Protein synthesis4	7.63	9.65	9.55	11.89	10.12	9.42	1.19	0.10	0.78	0.76	0.04	0.36
Protein degradation5	6.70	8.77	8.64	10.74	8.98	8.12	1.18	0.18	0.73	0.71	0.04	0.38
Hcys production6	2.95	3.12	3.59	1.85	2.87	2.63	0.99	0.49	0.28	0.90	0.69	0.75
Transsulfuration7	0.36	0.45	0.38	1.84	1.85	1.68	0.09	<0.01	0.35	0.19	0.23	0.88
Remethylation8	2.60	2.64	3.16	0.11	0.96	1.44	0.97	0.02	0.31	0.97	0.67	0.79

¹G-L, linear effect of GAA; G-Q, quadratic effect of GAA.

²Metabolizable Met = ((DM intake, kg/hr × 6.71 mmol metabolizable Met/kg DM intake [Campbell et al., 1997]) + supplemental Met.

³Met deposition = N retention, g/hr × 6.25 g protein/g N × 0.134 mmol Met/g protein (Ainslie et al., 1993).

⁴Protein synthesis = PD + Met deposition.

⁵Protein degradation = 1-¹³C-l-Met flux − metabolizable Met.

⁶Homocysteine production (use of Met in methylation reactions) = Methyl-²H₃-l-Met flux – PS.

⁷Transsulfuration = 1-¹³C-l-Met flux – PS.

⁸Remethylation = Methyl-²H₃-l-Met flux – 1-¹³C-l-Met flux.

Effects of Met

Supplemental Met increased (P < 0.01) 1-¹³C-l-Met flux (irreversible loss rate of the Met skeleton). A sizeable part of the 2.90 mmol/hr increase in Met flux can be accounted by the 1.68 mmol/hr of Met supplemented. In contrast, the methyl-²H₃-l-Met flux was not affected by supplementation of Met (P = 0.17).

Methionine supplementation increased metabolizable Met supply (P < 0.01; by experimental design) and Met deposition (P < 0.01; estimated from N retention). Protein synthesis tended to increase (P = 0.10) in response to Met supplementation, but PD was not significantly affected by Met supplementation. Because Met deposition was increased by Met supplementation, the difference between PS and PD was obligatorily increased.

Transmethylation reactions (i.e., homocysteine production) were not affected by Met supplementation, which is an interesting observation that suggests that transmethylation reactions may not have been limited by Met availability. Transsulfuration reactions were increased (P < 0.0001) with Met supplementation, whereas remethylation reactions decreased (P = 0.02) when Met was supplemented. The increase in transsulfuration reaction and reduction in remethylation reaction matches reasonably well with decreases in plasma serine and increases in plasma cysteine when supplemental Met was provided. Serine is required for the conversion of homocysteine to cysteine and thus it is frequently observed that Met supplementation to Met-deficient cattle leads to decreases in plasma serine (Titgemeyer and Merchen, 1990; Lambert et al., 2002). Elevated hepatic SAM inhibits activity of enzymes that catalyze homocysteine remethylation (Mato et al., 2008). Consequently, Met supplementation, which leads to elevated hepatic SAM content and to the inhibition of remethylation reactions, can shift the metabolism of homocysteine away from remethylation and toward transsulfuration.

Interactions between GAA and Met

There were no main effects of GAA on Met flux and related measures in Table 4, but there were interactions between GAA and Met, suggesting that the Met status of the cattle affected their response to the supplemental GAA. The whole-body flux of methyl-²H₃-l-Met increased in response to GAA provision when GAA was provided along with no supplemental Met, but, when steers received GAA along with 6 g/d Met, the flux of Met methyl groups was decreased by GAA provision (Table 4; Met × GAA-linear, P = 0.03). A Met × GAA-linear interaction (P = 0.04) was observed for flux of 1-¹³C-l-Met, which was increased by GAA supplementation when steers received no Met, but decreased by GAA supplementation when the steers received 6 g/d Met.

There were interactions between Met and GAA for both PS and PD (Met × GAA-linear; P = 0.04), where both measures followed a pattern similar to those observed for 1-¹³C-l-Met and methyl-²H₃-l-Met. Specifically, both PS and PD increased in response to GAA supplementation when Met was not supplemented, but decreased in response to GAA supplementation when 6 g/d Met was supplemented. Because PS and PD followed a similar pattern in response to treatments, the net effect was that protein deposition yielded a response different than that for PS. Deposition of Met was not statistically affected by GAA supplementation. The lack of an interaction between Met and GAA for Met deposition is in contrast to the observation discussed above that N retention tended (P = 0.10) to increase in response to supplemental GAA when Met supply was adequate. Although the pattern for Met deposition was similar to that for N retention, the lack of any statistical effect for Met deposition may be attributed to observations that were missing from the flux measurements, which reduced statistical power.

In our experiment, GAA supplementation had no significant effect on methylation reactions (homocysteine synthesis), transsulfuration, or remethylation, and no interactions between GAA and Met for methylation, transsulfuration, and remethylation reactions were detected. If all of the supplemental GAA (up to 5.3 mmol/hr) were methylated to creatine, the 1.68 mmol/hr (6 g/d) of supplemental Met would be inadequate for completely methylating the GAA. Moreover, our results showed that total methylation reactions were not increased by supplemental GAA. Thus, if the supplemental GAA was methylated, then the rate of other transmethylation reactions, such as choline synthesis, would have to correspondingly decrease for total methylation reactions to be unaffected by the GAA supplementation. In agreement with this suggestion, McBreairty et al. (2013, 2015), working with Yucatan miniature pigs receiving GAA through venous infusion or diet, demonstrated the increased methyl demand created by GAA use for creatine synthesis led to reductions in phosphatidylcholine synthesis.

Plasma AA

The effect of GAA and Met supplementation on plasma AA is shown in Table 5. As expected, Met supplementation increased plasma Met concentrations (P < 0.001), but plasma Met was not affected by GAA treatment. Our response in plasma Met concentrations agrees with our previous work (Ardalan et al., 2020) which suggested that methyl group deficiency induced by administration of GAA was not severe enough to alter plasma Met concentrations. Li et al. (2020) and Liu et al. (2020, 2021) also observed no effect of GAA administration to Angus bulls on plasma Met concentrations.

Table 5.

Effect of Met and GAA supplementation on plasma AA concentrations from blood collected on day 10

	0 Met			6 g/d Met
	GAA, g/d							P-value 1
AA, µM	0	7.5	15	0	7.5	15	SEM	Met	G-L	G-Q	Met × G-L	Met × G-Q
n	7	7	7	7	6	6
Homocysteine	11.6	13.7	12.7	9.9	9.9	10.0	1.22	<0.01	0.26	0.10	0.36	0.10
Methionine	12.7	12.5	14.4	23.8	22.6	25.2	1.72	<0.01	0.21	0.19	0.93	0.67
Cysteine	92.8	98.3	95.8	109.7	113.5	110.4	4.82	<0.01	0.40	0.06	0.59	0.89
Ornithine	77.6	62.4	73.0	54.3	59.8	59.9	6.37	0.03	0.93	0.28	0.35	0.11
Tryptophan	34.0	30.7	35.3	28.3	30.0	30.3	2.38	0.06	0.48	0.43	0.87	0.25
Tyrosine	45.1	47.7	51.2	37.7	45.1	43.4	4.91	0.09	0.16	0.57	0.96	0.49
Threonine	120.8	129.7	112.8	96.0	124.2	128.3	16.20	0.54	0.14	0.09	0.02	0.95
Leucine	155.6	126.0	144.6	106.9	106.8	110.2	10.3	<0.01	0.60	0.05	0.33	0.09
Isoleucine	119.7	107.4	115.5	94.9	96.6	98.5	5.70	<0.01	0.95	0.22	0.41	0.22
Valine	332.4	290.6	308.8	241.1	257.2	254.8	19.3	<0.01	0.67	0.32	0.12	0.07
Lysine	120.0	102.9	123.5	89.3	95.2	106.8	8.46	0.02	0.23	0.16	0.42	0.29
Phenylalanine	61.4	56.0	60.1	58.6	58.3	62.1	5.73	0.90	0.82	0.44	0.63	0.76
Glutamic acid	139.9	155.0	143.4	156.4	149.4	160.6	17.33	0.58	0.71	0.81	0.97	0.22
Glutamine	201.7	196.0	232.4	191.0	224.6	202.8	21.61	0.75	0.17	0.80	0.53	0.08
Aspartic acid	4.9	5.7	5.8	5.7	6.3	6.2	0.53	0.33	0.10	0.35	0.54	0.94
Asparagine	29.1	31.5	32.2	28.5	33.0	32.0	2.42	0.90	0.12	0.32	0.91	0.60
Proline	57.7	56.8	59.7	53.5	57.7	55.8	2.55	0.27	0.42	0.80	0.94	0.29
Alanine	193.7	218.8	207.1	199.1	225.1	219.0	11.64	0.41	0.16	0.10	0.78	0.90
Serine	174.0	149.2	169.6	68.9	138.3	89.7	17.09	<0.01	0.60	0.19	0.42	0.01
Glycine	229.5	243.4	223.5	178.8	231.0	193.7	23.92	0.06	0.81	0.07	0.58	0.40

¹G-L, linear effect of GAA; G-Q, quadratic effect of GAA.

Plasma concentrations of homocysteine were reduced (P < 0.01) in response to supplemental Met. Supplementation of GAA tended to increase plasma homocysteine in steers receiving no Met quadratically (the intermediate amount of GAA increased homocysteine the most), but GAA did not increase plasma homocysteine in steers receiving supplemental Met (Met × GAA-quadratic; P = 0.10). The lack of an increase of plasma homocysteine concentrations in response to supplemental GAA in the presence of supplemental Met could show a role of Met in providing enough methyl groups to prevent a methyl group deficiency. Therefore, administration of Met, as a methyl donor, reduced homocysteine levels (Zhou et al., 2016). These results agree with our previous research (Ardalan et al., 2020) conducted on GAA supplementation to dairy heifers; heifers receiving 12 g/d of Met demonstrated no increase in plasma homocysteine in response to GAA supplementation. In contrast, the heifers receiving no supplemental Met showed elevated concentrations of plasma homocysteine when either 30 or 40 g/d of GAA was provided. In our previous research (Ardalan et al., 2020), plasma homocysteine was increased 20% with GAA supplementation up to 30 g/d (0.06 g GAA/kg BW daily) when no Met was provided, whereas in the current experiment an 8% increase in plasma homocysteine concentrations was observed when 15 g/d of GAA (0.09 g GAA/kg BW daily) was supplemented without Met. The increases in plasma homocysteine concentrations in response to GAA infusions with no supplemental Met would suggest that a methyl group deficiency was generated by increasing the consumption of methyl groups for creatine synthesis from GAA. As noted previously, this could result in reduced availability of methyl groups for transmethylation reactions other than creatine production (e.g., choline synthesis, McBreairty et al., 2015).

In studies conducted on Angus bulls receiving GAA, Li et al. (2020) and Liu et al. (2020, 2021) demonstrated no differences in blood homocysteine when GAA was supplemented. We speculate that this lack of change in blood homocysteine reflects that the cattle were not deficient in Met supply, which agrees with our responses when 6 g/d of Met was supplemented. The lack of change in plasma homocysteine in those studies also could be the result of the relatively low amount of GAA that was fed (0.6 g GAA/kg diet DM).

Plasma homocysteine is considered a good marker for methyl group deficiency (da Costa et al., 2005; Setoue et al., 2008). Work with rats (Stead et al., 2001; Fukada et al., 2006) and humans (Ostojic et al., 2014, 2013a) demonstrated increases in plasma homocysteine when GAA was supplemented, and Stead et al. (2001) showed that rat hepatocytes treated with GAA had increased export of homocysteine. Thus, elevation of plasma homocysteine concentration after treatment with GAA can be linked to the increased demand for methyl groups to convert GAA to creatine (Fukada et al., 2006). In rodents, the hyperhomocysteinemia induced by GAA can be suppressed by supplementation with choline or betaine as sources of methyl groups (Setoue et al., 2008). Hence, supplemental methyl group sources such as Met can play an important role in preventing homocysteine accumulation by increasing homocysteine remethylation or by increasing the flux of homocysteine through transsulfuration (Zhou et al., 2016). Data from our flux measurements (Table 4) demonstrated that transsulfuration was increased by Met supplementation, whereas homocysteine remethylation was reduced; these changes reflect the role of Met not only as a methyl donor but also as an important regulator of Met-regulating reactions (Finkelstein and Martin, 1986).

Plasma cysteine concentrations were increased by Met supplementation (P < 0.01). This observation is consistent with the greater fluxes of Met through transsulfuration when Met was supplemented (Table 4). Under normal physiological conditions, the balance between the homocysteine production and elimination maintains the homocysteine balance in the body. An animal’s methyl group status is a critical determinant of the partitioning of homocysteine between transsulfuration (irreversible degradation to cysteine) and remethylation (recycling to Met) pathways. Overall, when dietary intake of Met is sufficient and Met does not need to be conserved, excessive intake of Met leads to decreased remethylation of homocysteine and increased transsulfuration of homocysteine (Nygard et al., 1999); lower availability of Met diverts homocysteine away from transsulfuration and consequently increases remethylation (Blom and Smulders, 2011).

Plasma cysteine concentrations increased quadratically (P = 0.06) with GAA supplementation with the greatest increase resulting from the intermediate amount of GAA, although differences were not particularly large. Although there was no effect of GAA on transsulfuration (Table 4), it is possible differences in transsulfuration were too small to detect statistically, yet large enough to modestly affect plasma cysteine concentrations. It is also possible that factors other than production rate of cysteine were affecting plasma cysteine concentrations.

Plasma concentrations of serine were decreased (P = 0.001) in response to Met supplementation. Decreased serine concentration in response to Met treatment could reflect the use of serine to produce cystathionine during Met transsulfuration (Awawdeh et al., 2004). Finkelstein and Martin (1986) observed an increase in the hepatic cystathionine synthase activity and a decrease in the concentrations of serine in liver of rats receiving Met. The authors suggested that increased activity of cystathionine synthase led to increased consumption of serine and homocysteine to synthesize cystathionine, which consequently results in decreased serine concentration. Also, a Met × GAA-quadratic interaction (P = 0.01) was observed for serine, because 7.5 g/d GAA decreased plasma serine of steers receiving no Met, but increased plasma serine for those steers supplemented with Met; we have no explanation for this effect.

Methionine supplementation decreased (P < 0.05) plasma concentrations of BCAA and lysine. The decreases in plasma BCAA in response to Met supplementation may have been due to increased uptake and utilization of BCAA for protein accretion when supplemental Met was provided (Campbell et al., 1997).

Plasma concentrations of ornithine were decreased (P < 0.03) when Met was supplemented, whereas plasma ornithine was not affected by supplemental GAA. In previous work (Ardalan et al., 2020), increases were observed for plasma arginine and ornithine for heifers receiving GAA supplementation, suggesting the arginine-sparing effect of GAA supplementation increased arginine availability, which could lead to more ornithine being formed from arginine. We did not measure plasma arginine in the current experiment, but, if arginine was spared by GAA supplementation, the effect did not carry through to affect plasma ornithine concentrations.

Conclusions

Increases in plasma creatine in association with no increase in urinary excretion of GAA in response to postruminal supplementation of GAA suggests that GAA can be an effective way to increase creatine availability to cattle. This experiment demonstrated that GAA supplementation may improve protein deposition when methyl group sources such as Met are supplemented concurrently or provided in adequate amounts by the basal diet. Because supplemental GAA elevated plasma homocysteine, the experimental model may be useful for inducing a methyl group deficiency in cattle. Supplementation of GAA did not significantly increase methylation reactions, suggesting that methylation reactions other than GAA synthesis may have been reduced by GAA supplementation. Further research is necessary to elucidate and fully understand the nature and extent of these alterations following GAA provision to cattle.

Glossary

Abbreviations

AA

amino acid

BCAA

branched-chain amino acids

BW

body weight

DM

dry matter

GAA

guanidinoacetic acid

HPLC

high-performance liquid chromatography

PD

protein degradation

PS

protein synthesis

SAM

S-adenosylmethionine

Contribution no. 21-212-J from the Kansas Agricultural Experiment Station. This work was supported by the State of Kansas through the Kansas State University Global Food Systems Innovation Program and by the USDA National Institute of Food and Agriculture, Hatch project 1001435.

Conflict of Interest Statement

The authors declare no real or perceived conflicts of interest.