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Ruminal ammonia load affects leucine 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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Six ruminally cannulated Holstein steers (initial BW = 189 ± 11 kg) housed in metabolism crates were used in a 6 x 6 Latin square to study effects of ruminal ammonia load on Leu utilization. All steers received a diet based on soybean hulls (2.7 kg of DM/d), ruminal infusions of 200 g of acetate/d, 200 g of propionate/d, and 50 g of butyrate/d, as well as an abomasal infusion of 300 g of glucose/d to provide energy without increasing microbial protein supply and an abomasal infusion of a mixture (238 g/d) of all essential AA except Leu. Treatments were arranged as a 3 x 2 factorial and included Leu (0, 4, or 8 g/d) infused abomasally and urea (0 or 80 g/d) infused ruminally. Abomasal Leu infusion linearly decreased (P < 0.05) both urinary and fecal N excretions and linearly increased (P < 0.05) retained N, but the decreases in urinary N excretion in response to Leu tended (P = 0.07) to be greater, and the increases in retained N in response to Leu were numerically greater in the presence of the urea infusion. Although urea infusions increased (P < 0.05) plasma urea concentrations, urinary N excretions, and urinary urea excretions, retained N also was increased (P < 0.05). The efficiency of deposition of supplemental Leu ranged from 24 to 43% when steers received 0 or 80 g of urea/d, respectively. Under our experimental conditions, increasing ammonia load improved whole-body protein deposition in growing steers when Leu supply was limiting.

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

	Introduction

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In the liver, absorbed ammonia (NH₃) is detoxified mainly to urea. Some researchers suggested that AA N might be used to support ureagenesis for NH₃ detoxification because NH₃ did not account for all of the released urea when ammonium salts were mesenterically infused in sheep and cattle (Symonds et al., 1981; Barej et al., 1987; Orzechowski et al., 1988). This conflicts reports in rat and sheep liver that NH₃ can contribute both N atoms for ureagenesis (Cooper et al., 1987; Luo et al., 1995; Brosnan et al., 1996). The use of AA N in ureagenesis may affect the use of AA for protein synthesis. In fact, NH₃ loading increased oxidation of Leu in vivo (Lobley et al., 1995) and Met and Ala in vitro (Mutsvangwa et al., 1996, 1999).

Ruminal NH₃ loading had no negative effects on N retention by sheep and cattle (Norton et al., 1982; Moorby and Theobald, 1999), nor on the oxidation of Ala, Glu, Leu, or Phe in vitro (Mutsvangwa et al., 1996, 1997, 1999), suggesting little or no effect of NH₃ loading on AA utilization (Lobley et al., 1996).

Ammonia loading led to nonsignificant improvements in N retention by growing steers limited by His (McCuistion et al., 2004), but Awawdeh et al. (2004) observed no effect of NH₃ loading in growing steers limited by Met. Thus, the effect of NH₃ loading on AA utilization may depend on the AA being studied. Because of the differences in the metabolic pathways of His, Met, and Leu, we hypothesized that NH₃ loading could yield different responses when Leu was limiting. For example, Leu is catabolized throughout the body, rather than principally in the liver, and the initial catabolic step for Leu is transamination. Moreover, Leu was a limiting AA under our experimental conditions (Löest et al., 2001), so effects of NH₃ loading, if present, likely could be demonstrated. Our objective was to study the effects of ruminal NH₃ loading on Leu utilization by growing steers.

	Materials and Methods

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

Six ruminally cannulated Holstein steers (189 ± 11 kg) fitted with ruminal and abomasal infusion lines were used in a 6 x 6 Latin square design to study the effects of ruminal NH₃ load on Leu utilization. Steers were housed in individual metabolism crates in a temperature-controlled room (21°C) under continuous lighting.

Before initiation of 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 2.7 kg of DM/d 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 AA to the small intestine. All steers received continuous ruminal infusions of 200 g of acetate/d, 200 g of propionate/d, and 50 g of butyrate/d as well as abomasal infusions of 300 g of glucose/d 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), that supplied nonessential and all essential AA except Leu to ensure that Leu was the most-limiting AA for N retention. Leucine was added to the AA mixture according to treatment.

Treatments were arranged as a 3 x 2 factorial and included three amounts of L-Leu (0, 4, or 8 g/d) infused continuously into the abomasum and two amounts of urea (0 or 80 g/d) infused continuously into the rumen. Each experimental period lasted for 6 d (2 d for adaptation to treatment and 4 d for total fecal and urinary collection). The 2-d adaptation periods are adequate because ruminants rapidly adapt to changes in nutrients supplied postruminally (Hovell et al., 1983; Moloney et al., 1998).

Abomasal infusate for each steer was prepared by dissolving L-Ile and L-Val in 1 kg of water containing 60 g of 6 M HCl. Once L-Ile and L-Val were dissolved, the remaining AA, except Glu, were added to the mixture. Glutamate was dissolved separately in 500 g of 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 Lambert et al. (2004) demonstrated that steers maintained under our experimental conditions were deficient in one or more of these vitamins. Leucine was dissolved separately and added to the mixture according to treatment (0, 4, or 8 g/d). Ruminal infusates for each steer were prepared by mixing 200 g of acetate/d, 200 g of propionate/d, and 50 g of butyrate/d. 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 or 80 g/d).

Representative samples of the basal diet for each period were collected daily, stored (–20°C), and ground (1-mm screen Wiley mill) before analysis. Orts, if any, were collected on d 2 through 5, composited, stored (–20°C), and ground before 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 analyses.

Samples of the diet, orts, and feces were analyzed for DM (105°C in a forced-air oven for 24 h) and OM (weight loss after ashing at 450°C for 8 h) to calculate digestibility. Composite samples of the diet, orts, wet feces, and urine were analyzed for N using LECO FP 2000 Nitrogen Analyzer (LECO Corporation, St. Joseph, MI), and data were used 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 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, as well as concentrations of serum insulin and IGF-I.

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

Plasma glucose concentration was 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 were measured using 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). Serum IGF-I was measured using an active IGF-1 coated-tube two-site immunoradiometric assay kit (DSL-5600; Diagnostic Systems Laboratories).

Nitrogen balance and plasma metabolite data were analyzed using the Mixed procedure of SAS System for Windows Release 8.1 (SAS Inst., Inc., Cary, NC). The model contained the effects of Leu, urea, Leu x urea, and period. Steer was included as a random variable. Linear and quadratic effects of Leu and their interactions with urea were tested using single df contrasts. Treatment means were computed using the LSMEANS option. Average ruminal NH₃ concentrations for each steer were analyzed using the Mixed procedure of SAS with level of urea supplementation as the sole factor evaluated.

	Results and Discussion

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Ruminal NH₃ concentrations averaged 9.0 mM for steers receiving 0 g of urea/d and 27 mM for those receiving 80 g of urea/d (P < 0.01). In vitro (Slyter et al., 1979) and in vivo (Satter and Slyter, 1974) studies showed that microbial synthesis 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 ruminal NH₃ concentrations in our study were adequate to support microbial requirements and that treatments did not affect ruminal fermentation. The increase (P < 0.05) in ruminal NH₃ concentrations and plasma urea (Table 4) in response to urea infusions indicates that urea infusions increased NH₃ absorption and that ruminal NH₃ loading was achieved.

Nitrogen retention linearly increased (P < 0.05) with abomasal Leu supplementation as a result of decreases (P < 0.05) in both urinary and fecal N excretions. The decrease in urinary N excretion in response to Leu tended to be greater when urea was supplemented (linear Leu x urea interaction; P = 0.07). Also, abomasal supplementation with Leu linearly decreased (P < 0.05) urinary urea N excretions, although the decreases in response to Leu were predominantly observed when 80 g of urea/d were supplemented (linear Leu x urea interaction; P < 0.05). The decreases in urinary urea N excretion in response to Leu supplementation were slightly greater than the decreases in total urinary N. Although the Leu x urea interaction was not significant for N retention (linear Leu x urea interaction; P = 0.53), the response mirrored the responses described previously for urinary N and urinary urea N in that the increases in N retention in response to Leu supplementation were greater when urea was supplemented than when it was not.

Using similar models, Greenwood and Titgemeyer (2000) reported that at least one of the branched-chain (BC) AA was limiting for N retention, and Löest et al. (2001) observed that Leu and Val were both limiting in the basal diet. Thus, the increase in retained N in response to abomasal supplementation of Leu in our study was an expected result.

The observed linear responses to Leu in N retention suggest that steer requirements for supplemental Leu are clearly > 4 g/d and, at least, not greatly < 8 g/d under our experimental conditions. Considering Leu supply from the basal diet, the total requirement for maximal N retention was likely near 24 g of Leu/d (calculated as 16 g of Leu/d provided from the basal diet [5.9 g of metabolizable Leu/kg of DMI; Campbell et al., 1997] plus 8 g of Leu/d provided via abomasal infusion). Although significant linear increases in plasma Leu concentrations (Table 4) were observed in response to Leu supplementation, the small magnitude of increase further supports the hypothesis that 4 g of supplemental Leu/d was clearly below the requirement.

Urea infusions increased (P < 0.05) N intake, retained N, total urinary N excretions, and urinary urea N excretions (Table 3). Fecal N excretions were not affected by urea infusions (P = 0.58; Table 3). Approximately 98, 81, and 76% of the additionally infused urea N was excreted as urinary urea N when 0, 4, or 8 g of Leu/d were supplemented, respectively. The increases in urinary urea N in response to NH₃ loading reflect most of the increases in total urinary N excretions. Of the additional increase in total urinary N, 96% was excreted as urea N.

The increase in retained N with urea infusions is in contrast to our initial hypothesis that NH₃ loading might decrease N retention by increasing catabolism of the limiting AA (Leu), but the response is unlikely to be related to changes in ruminal fermentation because there was no effect (P 0.80) of urea infusion on dietary DM or OM digestibility. Leucine metabolism occurs throughout the body (Harper et al., 1984), so changes in catabolism in response to NH₃ loading could have occurred in either the liver or in the extrahepatic tissues. Increasing the NH₃ supply to the liver may indirectly alter Leu catabolism throughout the body by altering the amount of substrates such as Gln and Ala that are available to extrahepatic tissues. Lobley et al. (1996) demonstrated that when NH₃ loading was imposed in sheep, hepatically removed NH₃ was used to synthesize AA (Asp, Glu, Gln). The BC AA are reversibly transaminated to BC -keto acids as a result of the action of BC AA transaminase. The amino group can be transferred to pyruvate and Glu, producing Ala and Gln, respectively (Harper et al., 1984). Thus, it is possible that the observed increase in retained N in response to NH₃ loading in our study was a result of decreasing the rate of BC AA transamination or increasing the rate of conversion of -ketoisocaproate to Leu through transamination reactions, or both, by altering the substrate available for these reactions. In fact, blood Leu concentrations increased in response to NH₃ loading in sheep (Milano et al., 2000). Although plasma concentrations of both Ala and Glu were numerically decreased, and those of Gln increased, by urea infusions (Table 4), it is unknown whether similar changes occurred in tissues throughout the body. Additionally, it should be noted that BC -keto acid dehydrogenase is considered the rate-limiting step in catabolism of Leu (Harper et al., 1984; Block, 1989).

Assuming that retained N was deposited completely as tissue protein (retained N x 6.25) and that the protein of tissue gain contains 6.7% Leu (Ainslie et al., 1993), the calculated efficiency of supplemental Leu utilization (between 0 and 4 g of supplemental Leu/d) was 24 and 43% for steers receiving 0 or 80 g of urea/d, respectively. The utilization efficiency calculated between 0 and 8 g of supplemental Leu/d was 24 and 35% for steers that received 0 or 80 g of urea/d, respectively. The slightly lower efficiency when calculated between 0 and 8 g of Leu/d may reflect that the steers’ requirement was slightly < 8 g of supplemental Leu/d. The apparently higher efficiency of Leu utilization in the presence of the urea infusion suggests that the NH₃ load decreased catabolism of Leu, the limiting AA in our study.

There were no significant effects of treatments on plasma glucose (P 0.10) or on serum insulin (P 0.42) or IGF-1 (P 0.27) concentrations (Table 4). Abomasal Leu supplementation linearly increased (P < 0.05) the plasma concentrations of Leu and linearly decreased (P < 0.05) the concentrations of Ala, Val, Ile, Pro, ornithine, and Tyr (Table 4). Similarly, abomasal supplementation of Leu decreased plasma concentrations of Tyr and Ala in growing steers (Greenwood and Titgemeyer, 2000; Löest et al., 2001). Löest et al. (2001) also demonstrated decreases in plasma concentrations of Ile, Tyr, and Val in response to Leu supplementation in growing steers. The decreases in Ala, Ile, Pro, Tyr, and Val were probably a result of increases in uptake and utilization of these AA for protein synthesis as Leu, the limiting AA under our experimental conditions, became available in greater amounts.

The decreases in plasma Ile and Val in response to supplemental Leu also might be a result of alterations in their metabolism. Branched-chain -ketoacid dehydrogenase, the rate-limiting enzyme for oxidative decarboxylation of -ketoacids during BC AA catabolism, is regulated predominantly by -ketoisocaproate, which depends on Leu supply (Block, 1989). Thus, as Leu was supplemented, it is likely that the activity of BC -ketoacid dehydrogenase increased, which might have contributed to reduced concentrations of Val and Ile.

Urea infusion decreased (P < 0.05) the plasma concentrations of Ala, Asp, and Phe. A similar effect for Ala in response to urea infusions was observed in our laboratory when Met supply was limiting (Awawdeh et al., 2004). The decrease in plasma Ala may reflect increased Ala oxidation as was observed in hepatocytes isolated from sheep fed high-urea diets (Mutsvangwa et al., 1996). Moreover, Ala may be used as a source of Asp N for detoxification of NH₃ load. In the urea cycle, Asp is one substrate that provides N for urea synthesis. Thus, the observed decrease in plasma Asp might be a result of the increased demands of Asp for urea synthesis.

Plasma concentrations of Glu tended to decrease (P = 0.08) and those for Gln tended to increase (P = 0.08) in response to urea infusions (Table 4). These changes could be due to the amidation of Glu to Gln as a secondary means of hepatic NH₃ detoxification (Häussinger et al., 1992). Similar trends for decreased plasma Glu and increased Gln in response to NH₃ loading were observed in growing steers when either His (McCuistion et al., 2004) or Met (Awawdeh et al., 2004) was the AA most limiting for animal performance, which supports our proposed hypothesis regarding Glu and Gln. In agreement with our observation regarding Glu and Gln, abomasal infusion of NH₄HCO₃ in sheep increased hepatic Gln production (Nieto et al., 2002). Milano and Lobley (2001) suggested that NH₃ loading via mesenteric infusion of NH₄HCO₃ in sheep stimulated Gln synthesis in tissues other than the liver.

We studied the effects of NH₃ load under conditions in which Leu supply was limiting. To achieve that, the diet was formulated to provide insufficient quantities of AA to the small intestine, and all essential AA, except Leu, were supplemented. Leucine was selected in this study because it was demonstrated as one of the most limiting AA under our experimental conditions (Löest et al., 2001). If Leu supply was not limiting, effects of NH₃ loading on Leu utilization might not lead to any changes in performance because an excess supply of Leu could allow for optimal performance even in the face of changes in efficiency of use. It was reported that NH₃ 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 in which it was clear that AA supply limited performance.

Ammonia loading improved N retention and the efficiency of utilization of supplemental Leu by growing steers. McCuistion et al. (2004) observed that N retention was not significantly affected by NH₃ loading in growing steers when His supply limited animal performance, although numerically the NH₃ loading improved N retention. Awawdeh et al. (2004) observed no effect of NH₃ loading in growing steers limited by Met. Thus, the effect of NH₃ loading seems to depend on which AA is being studied.

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 Leu. The data of Lobley et al. (1995) demonstrated that an NH₃ load increased oxidation of Leu in sheep, although this response has been attributed to metabolic acidosis in response to NH₄Cl infusion (Lobley et al., 1996). Our results also contrast the data of Mutsvangwa et al. (1999), in which Met deamination was increased with NH₃ loading in vitro.

The utilization efficiency of supplemental Leu was lower than the 65% efficiency value predicted by Ainslie et al. (1993) and used for estimating AA requirements in NRC (1996). The NRC (1996) assumes the same utilization 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 utilization of supplemental AA has been noted previously for Met (Campbell et al., 1996; Lambert et al., 2002). Recently, McCuistion et al. (2004) observed an efficiency of utilization for supplemental His (65%) that was greater than our observation for Leu. The efficiency of His utilization was close to the value predicted by NRC (1996), suggesting that there are differences among AA in how efficiently they are used by cattle. In addition, our data suggest that, at least for Leu, the efficiency may depend on the nutritional status of the animal.

	Implications

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Under our experimental conditions, ruminal ammonia loading did not lead to metabolic costs with regard to leucine utilization for protein deposition. In fact, increasing the ruminal ammonia concentration above the recommended levels required to optimize ruminal fermentation improved the whole animal protein deposition when leucine supply was limiting. However, at present, environmental and economic costs may not justify the use of ammonia loading as a means of improving cattle performance.

	Footnotes

 
¹ Contribution No. 04-287-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 August 3, 2004. Accepted for publication January 10, 2005.

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