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Effects of ammonia load on methionine utilization by growing steers¹

M. S. Awawdeh^*, E. C. Titgemeyer^*,2, K. C. McCuistion^* and D. P. Gnad

^* Department of Animal Sciences and Industry and and Department of Clinical Sciences, Kansas State University, Manhattan 66506-1600

	Abstract

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Seven ruminally cannulated Holstein steers (194 ± 16 kg) housed in metabolism crates were used in a 6 x 6 Latin square, with one additional steer, to study effects of ruminal ammonia load on methionine (Met) use. All steers received a diet based on soybean hulls (2.6 kg DM/d), ruminal infusions of 200 g/d of acetate, 200 g/d of propionate, and 50 g/d of butyrate, as well as abomasal infusion of 300 g/d of glucose to provide energy without increasing microbial protein supply, and abomasal infusions of a mixture (248 g/d) of all essential AA except Met. Treatments were arranged as a 3 x 2 factorial and included urea (0, 40, or 80 g/d) infused ruminally to supply metabolic ammonia loads and Met (2 or 5 g/d) infused abomasally. Supplementation with the greater amount of Met decreased (P < 0.05) urinary N excretion from 68.8 to 64.8 g/d and increased (P < 0.05) retained N from 22.0 to 27.5 g/d. Urea infusions linearly increased (P < 0.05) urinary N excretions, plasma urea concentrations, and urinary urea excretions, but retained N was not affected. The efficiency of deposition of supplemental Met, calculated by assuming that Met deposition is 2.0% of protein deposition (6.25 x retained N), ranged between 18 and 27% when steers received 0 or 80 g/d of urea, respectively. There were no (P 0.40) effects of treatments on serum insulin or IGF-I concentrations. In our model, increasing ammonia load did not affect whole-body protein deposition in growing steers when Met was limiting.

Key Words: Amino Acids • Ammonia • Cattle • Growth • Methionine • Utilization

	Introduction

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Ammonia generated from the degradation of protein and other nitrogenous compounds in the rumen is transported to the liver, where it is predominantly detoxified to urea. Some studies indicate that AA N is required for ureagenesis, and this may contribute to inefficient use of dietary AA. Urea N released by the liver exceeded the hepatic uptake of NH₃ when ammonium salts were infused into the mesenteric vein of sheep (Barej et al., 1987; Orzechowski et al., 1988) and cattle (Symonds et al., 1981; Wilton et al., 1988), suggesting that additional N sources, such as AA, are required for NH₃ detoxification (Lobley et al., 1995). Ammonia loading increased leucine oxidation in vivo (Lobley et al., 1995) and methionine (Met) deamination in vitro (Mutsvangwa et al., 1999). Furthermore, hepatocytes isolated from sheep fed diets containing excess ruminally available N demonstrated greater alanine oxidation than those from sheep fed a more typical diet (Mutsvangwa et al., 1996, 1997).

However, other studies demonstrated no obligatory need of AA N for urea synthesis. Challenging ovine hepatocytes with NH₄Cl in vitro had no effect on alanine, glutamate, leucine, or phenylalanine oxidation (Mutsvangwa et al., 1996, 1997, 1999), and NH₃ contributed both N atoms for ureagenesis in rat and sheep liver (Cooper et al., 1987; Luo et al., 1995; Brosnan et al., 1996). Furthermore, ammonia loading in sheep had no negative effects on N retention (Norton et al., 1982), nor on the hepatic uptake of total, essential, or branched-chain AA (Milano et al., 2000; Milano and Lobley, 2001), suggesting that NH₃-enhanced ureagenesis has no obligatory requirement for AA N (Lobley et al., 1996).

At this moment, the exact nature of the effect of ammonia loading on AA use has not been well assessed. Our objective was to study the effects of ruminal ammonia loading on Met use by growing cattle.

	Materials and Methods

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The Kansas State University Institutional Animal Care and Use Committee approved procedures involving animals in this study.

Seven ruminally cannulated Holstein steers (194 ± 16 kg initial BW) fitted with ruminal and abomasal infusion lines were used in a 6 x 6 Latin square to study the effects of ammonia load on Met use. The one additional steer was included in the study to increase the number of observations, and it was provided the same treatment sequence as one of the other steers. Four observations were not obtained due to failures in the collection of excreta. Steers were housed in individual metabolism crates in a temperature-controlled room (21°C) under continuous lighting.

Before starting the study, steers were adapted to the basal diet (Table 1) for 2 wk and to ruminal and abomasal infusions for 5 d. All steers had free access to water and received the same basal diet at 2.6 kg/d DM in equal proportions at 12-h intervals. The basal diet, which was characterized for metabolizable AA supply by Campbell et al. (1997), was formulated to provide adequate ruminally degraded protein but low amounts of metabolizable AA. All steers received continuous ruminal infusions of 200 g/d of acetate, 200 g/d of propionate, and 50 g/d of butyrate, as well as an abomasal infusion of 300 g/d of glucose to supply additional energy without increasing microbial protein supply. All steers received continuous abomasal infusions of an AA mixture (Table 2) as described by Greenwood and Titgemeyer (2000), which supplied nonessential and all essential AA, except Met, to ensure that Met was the most limiting AA for N retention. Methionine was added to the AA mixture according to treatment.

Abomasal infusate for each steer was prepared by dissolving the branched-chain AA (L-valine, L-leucine, and L-isoleucine) in 1 kg of water containing 60 g of 6 M HCl. Once the branched-chain AA were dissolved, the remaining AA, except glutamate, were added to the mixture. Glutamate was dissolved separately in 500 g water containing 30 g of NaOH. After all AA were dissolved, the two solutions of AA were mixed together, 300 g of glucose was added, and water was added to bring the total weight of the daily infusate to 4 kg. The pH of the infusate was 5.5. Pyridoxine•HCl (10 mg/d), folic acid (10 mg/d), and cyanocobalamin (100 µg/d) were added to the mixture because data demonstrated that steers maintained under our experimental conditions were deficient in one or more of these vitamins (Lambert et al., 2004). Methionine was dissolved separately in water and added to the mixture according to treatment (2 or 5 g/d).

Ruminal infusates for each steer were prepared by mixing 200 g/d of acetate, 200 g/d of propionate, and 50 g/d of butyrate. Water was added to bring the final weight of the mixture to 4 kg/d. Urea was added to the mixture according to treatment (0, 40, or 80 g/d). Infusion lines of flexible polyvinylchloride tubing (2.4 mm i.d.) were placed in the rumen and abomasum through the ruminal cannula. A perforated vial was attached to the end of the ruminal infusion lines to avoid direct infusion of VFA onto the ruminal wall. Rubber flanges (8-cm diameter) were attached to the end of the abomasal infusion lines to ensure that they remained in the abomasum. Solutions were continuously infused into the rumen and abomasum using a peristaltic pump.

Representative samples of the basal diet for each period were collected daily and stored (–20°C) for later analysis. Orts, if any, on d 2 through 5 were collected, composited, and stored (–20°C) for later analysis. Feces and urine for each steer were collected from d 3 through 6 of each period and weighed to determine the total output. Urine was collected in buckets containing 300 mL of 6 M HCl to prevent NH₃ loss. Representative samples of feces (10%) and urine (1%) were saved, composited by period, and stored (–20°C) for later analysis.

Samples of the diet, orts, and feces were analyzed for DM (105°C in forced-air oven for 24 h) and OM (weight loss on ashing at 450°C for 8 h) to calculate digestibilities. Composite samples of the diet, orts, wet feces, and urine were analyzed for N using a Leco FP 2000 nitrogen analyzer (Leco Corp., St. Joseph, MI) to calculate N retention.

Jugular blood samples were collected 4 h after the morning feeding on the last day of each period. Blood was collected into vacuum tubes (Becton Dickinson, Franklin Lakes, NJ) containing sodium heparin, immediately chilled on ice, and centrifuged for 20 min at 1,000 x g to obtain plasma. Blood also was collected into vacuum tubes without additives, allowed to clot for 30 min at room temperature, and then centrifuged for 20 min at 1,000 x g to obtain serum. Samples were stored (–20°C) for later analysis of plasma glucose, urea, and AA and of serum insulin and IGF-I.

After completion of the study, steers were maintained on their treatments for one more day, and ruminal fluid samples were collected at 3 and 6 h after morning feeding and stored (–20°C) for later analysis of NH₃.

Plasma glucose concentrations were measured using methods of Gochman and Schmitz (1972). Plasma and urinary urea were measured using the method of Marsh et al. (1965), and urinary and ruminal fluid NH₃ concentrations by the method of Broderick and Kang (1980). Plasma AA were measured by gas chromatography using a commercial kit (EZ:faast; Phenomenex, Torrance, CA). Insulin was measured using an insulin RIA kit (DSL-1600; Diagnostic Systems Laboratories, Webster, TX), and IGF-I was measured using an active IGF-I coated-tube IRMA kit (DSL-5600; Diagnostic Systems Laboratories).

Nitrogen balance and plasma metabolite data were analyzed statistically using the Mixed procedure of SAS (Release 8.1, SAS Inst., Inc., Cary, NC). The model contained the effects of Met, urea, Met x urea, and period. Steer was included as a random effect. Linear and quadratic effects of urea and their interactions with Met were tested using single degree of freedom contrasts. Treatment means were computed using the LSMEANS option. Ruminal NH₃ concentrations were analyzed using the Mixed procedure of SAS. The model contained the effects of time of sampling (3 or 6 h after infusions), level of urea supplementation, and urea supplementation x sampling time.

	Results and Discussion

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Ruminal NH₃ concentrations averaged 4.5, 21.5, and 19.7 mM for steers receiving 0, 40, and 80 g/d of urea, respectively (data not shown). The increases (P < 0.05) in ruminal NH₃ concentrations and plasma urea (see Table 4) in response to urea infusions indicate that urea infusions increased ammonia absorption and that ruminal ammonia loading was achieved. In vitro (Slyter et al., 1979) and in vivo (Satter and Slyter, 1974) studies showed that microbial growth was maximized when ruminal NH₃ concentrations were 1.6 and 3.5 mM, respectively. This, in addition to unchanged DM and OM digestibilities in response to urea infusion (Table 3), indicates that rumen NH₃ concentrations in our study were adequate to support microbial requirements and that treatments did not affect ruminal fermentation.

Although urea infusions linearly increased (P < 0.05) urinary N excretions from 48.5 to 67.3 and 84.5 g/d for steers infused with 40 and 80 g/d urea, respectively, retained N was not affected (P = 0.2) by urea treatments. Of the additional N infused as urea, 73 and 97% were excreted as urea N when 2 and 5 g/d Met was supplemented, respectively (Table 3). Urea infusions linearly increased (P < 0.05) urinary urea N (Table 3) from 38.9 to 54.5 and 70.2 g/d for the steers infused with 40 and 80 g/d of urea, respectively. These increases in urinary urea N in response to ammonia loading closely reflect the increase in total urinary N. Of the additional increases in total urinary N when 40 and 80 g/d of urea was infused, 83 and 87% were excreted as urea N, respectively.

To assess the efficiency of AA use, it is important that the input levels being tested are below animal requirements. Our levels of Met supplementation (2 and 5 g/d) were selected to be below the requirements of growing steers under our experimental conditions to ensure we were working in a range that provided a linear response to N retention with Met supplementation. Our diet (2.6 kg DM/d) provided 2.6 g/d of Met (Campbell et al., 1997), so even with the higher level of supplemental Met (5 g/ d) the total Met supply (7.6 g/d) was below the requirements of steers maintained under similar conditions (Campbell et al., 1997). Although plasma Met concentrations demonstrated a significant increase in response to the higher level of Met supplementation (Table 4), the observed increases were relatively small in magnitude and, in comparison to the greater increases in plasma Met observed by Campbell et al. (1997) in response to Met supplementation, suggest that 5 g/d of supplemental Met was below the steers’ requirement.

Assuming that retained N was deposited completely as tissue protein (retained N x 6.25) and that the protein of tissue gain contains 2.0% Met (Ainslie et al., 1993), the calculated efficiencies of use of supplemental Met (i.e., between 2 and 5 g/d of supplemental Met) were 23, 27, and 18% for steers receiving 0, 40, or 80 g/d of urea, respectively. Thus, our average efficiency of use of supplemental Met (23%) was similar to those reported by Campbell et al. (1996, 1997) and Titgemeyer and Merchen (1990), higher than those of 12 to 14% reported by Froidmont et al. (2000) and Lambert et al. (2002), but much lower than the 65% efficiency value predicted by Ainslie et al. (1993), which was adopted by the NRC (1996). Using a growth model, Wilkerson et al. (1993) estimated that Met requirements were 3.0% of metabolizable protein requirements; thus, if body protein contains 2.0% Met, the efficiency of Met use was approximately two-thirds as great for Met as for metabolizable protein as a whole.

The efficiencies of use of the basal Met supply (4.6 g/ d; calculated as 2.6 g/d provided from the diet [Campbell et al., 1997] plus 2 g/d provided to all steers via abomasal infusions) were much greater than those for the supplemental methionine. Nitrogen retention averaged 22.1 g/ d for steers receiving the lower level of infused methionine, and this would correspond to 2.8 g/d of Met being deposited by the steers at an efficiency of approximately 60%. Consideration of a maintenance requirement would increase this calculated efficiency. Although it seems that lesser supplies of Met are used more efficiently than greater ones, the efficiency of use of the basal Met supplies is likely overestimated due to the overestimation of protein deposition by N retention.

Our estimates of Met use do not consider the role of Met as a precursor for the synthesis of cysteine. In our experimental model, we have observed either no change in N retention in response to supplemental cysteine (Campbell et al., 1997; Löest et al., 2002) or only relatively small increases (Lambert et al., 2004). Thus, it does not seem advantageous to consider the role of Met as a precursor for cysteine synthesis; however, if cysteine production is considered as an important end product of Met, then the efficiencies of Met use could be nearly twice those calculated from our data.

The infusion of 40 g/d of urea, but not 80 g/d, decreased (quadratic; P < 0.05) plasma glucose concentrations (Table 4). The reasons for the decreased plasma glucose concentrations with the infusion of 40 g/d of urea are unknown, but the magnitude of change was small. Methionine supplementation did not affect plasma urea or glucose concentrations. Serum insulin and IGF-I were not affected by any treatment (Table 4).

Methionine supplementation increased (P < 0.05) plasma concentrations of Met and decreased (P < 0.05) concentrations of valine, leucine, serine, ornithine, and tyrosine (Table 4). The same trends were observed for valine, leucine, and serine in growing steers infused with L-Met (Titgemeyer and Merchen, 1990; Campbell et al., 1996, 1997) and similar trends were observed for tyrosine in growing steers infused with D-Met (Campbell et al., 1996). The decrease in plasma valine, leucine, and tyrosine concentrations might be a result of increased uptake and use of these AA for protein synthesis as Met, the first-limiting AA, became available in greater amounts. Serine is used in cystathionine synthesis during transsulfuration, and this may have been the reason for the observed decrease in plasma concentrations of serine in response to Met supplementation.

Plasma concentrations of glutamate and alanine were linearly (P < 0.05) decreased in response to urea infusions (Table 4). The decrease in plasma glutamate could be due to the amidation of glutamate to glutamine as a secondary means of hepatic NH₃ detoxification. Numerical increases in plasma glutamine concentrations in response to urea infusion were also observed, which supports this hypothesis. Trends for decreased plasma glutamate and increased glutamine in response to ammonia loading were similarly observed in growing steers when His was the AA most limiting for animal performance (McCuistion et al., 2004). The decrease in plasma alanine in response to urea infusion may reflect increased ala-nine oxidation, as was observed in hepatocytes isolated from sheep fed high-urea diets (Mutsvangwa et al., 1996). Moreover, alanine may be used as a source of aspartate N for detoxification of ammonia load.

We studied the effects of ammonia load under conditions where Met supply was limiting. To achieve that, the diet was formulated to provide deficient amounts of AA, and all essential AA, except Met, were supplemented. If protein (AA) supply were not limiting, negative effects of ammonia loading on AA use might not lead to any changes in performance because an excess supply of AA could allow for optimal performance even in the face of decreased efficiency of use. Ammonia loading had no negative effects on N retention in sheep (Norton et al., 1982) or cattle (Slyter et al., 1979; Moorby and Theobald, 1999), but those studies were not conducted under conditions where AA supply limited performance. Our data suggest that catabolism of Met is not impacted by increases in ureagenesis in response to an ammonia load.

Ammonia loading did not have negative effects on N retention or on the efficiency of use of supplemented Met by growing steers. McCuistion et al. (2004) similarly observed that N retention was not affected by ammonia loading in growing steers when histidine supply limited animal performance. Our results suggest that the additional urea synthesis to support NH₃ detoxification does not require an obligatory input of AA N, at least not from Met, as was suggested by the data of Mutsvangwa et al. (1999), where Met deamination was increased with ammonia loading in vitro. Our results also contrast with the data of Lobley et al. (1995), where ammonia loading increased leucine oxidation in sheep.

The efficiency of supplemental Met use was lower than the 65% efficiency value predicted by Ainslie et al. (1993) and used for estimating AA requirements by the NRC (1996). The NRC (1996) assumes the same use efficiency value for all AA, and the efficiency is based only on the equivalent BW of the animal. The overestimation by the NRC (1996) for efficiency of use of supplemental Met has been noted previously (Titgemeyer and Merchen, 1990; Campbell et al., 1996, 1997; Froidmont et al., 2000; Lambert, 2001). Recently, we have observed an efficiency of use for supplemental histidine greater than that for Met (65%; McCuistion et al., 2004), suggesting that there are differences among AA in how efficiently they are used by cattle.

	Implications

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Under our experimental conditions, ruminal ammonia loading did not lead to metabolic costs with regard to methionine use for growth. Although ammonia loading did not negatively affect whole-body protein deposition when methionine was limiting, excessive amounts of dietary protein may have environmental and economical costs that are generally considered unacceptable.

	Footnotes

 
¹ Contribution No. 04-019-J from the Kansas Agric. Exp. Stn., Manhattan. This research was supported by NRI Competitive Grants Program/CSREES/USDA, Award No. 2003-35206-12837.

² Correspondence: 132 Call Hall (e-mail: etitgeme{at}oznet.ksu.edu).

Received for publication February 3, 2004. Accepted for publication August 11, 2004.

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