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ANIMAL NUTRITION |
* Department of Animal Sciences and Industry and
and
Department of Clinical Sciences, Kansas State University, Manhattan 66506-1600
Abstract
Two experiments were conducted with ruminally cannulated Holstein steers to determine effects of N supply on histidine (His) utilization. All steers received 2.5 kg DM/d of a diet based on soybean hulls; abomasal infusion of 250 g/d amino acids, which supplied adequate amounts of all essential amino acids except His; abomasal infusion of 300 g/d glucose; and ruminal infusion of 180 g/d acetate, 180 g/d propionate, and 45 g/d butyrate. Both experiments were 6 x 6 Latin squares with treatments arranged as 3 x 2 factorials. No significant (P < 0.05) interactions between main effects were noted for N balance criteria in either Exp. 1 or 2. For Exp. 1, steers (146 ± 7 kg) received 0, 1.5, or 3 g/d of L-His infused abomasally in combination with 0 or 80 g/d urea infused ruminally to supply a metabolic ammonia load. Urea infusions increased (P < 0.05) ruminal ammonia concentration from 8.6 to 19.7 mM and plasma urea from 2.7 to 5.1 mM. No change in N retention occurred in response to urea (35.1 and 37.1 g/d for 0 and 80 g/d urea, respectively, P = 0.16). Retained N increased linearly (P < 0.01) with His (31.5, 37.8, and 39.0 g/d for 0, 1.5, and 3 g/d L-His, respectively). Efficiency of deposition of supplemental His between 0 and 1.5 g/d averaged 65%. In Exp. 2, steers (150 ± 6 kg) were infused abomasally with 0 or 1 g/d of L-His in combination with no additional amino acids (Control), 100 g/d of essential + 100 g/d of nonessential amino acids (NEAA+EAA), or 200 g/d of essential amino acids (EAA). Retained N increased (P = 0.02) from 34.2 to 38.3 g/d in response to His supplementation. Supplementation with NEAA+EAA increased (P < 0.05) N retention (33.9, 39.3, and 35.6 g/d for Control, NEAA+EAA, and EAA, respectively), likely in response to increased energy supply. Plasma urea concentrations of steers receiving NEAA+EAA (3.8 mM) and EAA (3.8 mM) were greater (P < 0.05) than those of Control steers (2.7 mM). The average efficiency of His utilization was 63%, a value similar to the value of 65% observed in Exp. 1, as well as the 71% value predicted by the Cornell net carbohydrate and protein system model. Under our experimental conditions, increases in N supply above requirements, as either ammonia or amino acids, did not demonstrate a metabolic cost in terms of His utilization for whole-body protein deposition by growing steers.
Key Words: Amino Acids • Cattle • Histidine • Utilization
Introduction
Histidine (His) has been identified as a limiting amino acid (AA) for growing cattle (Chalupa et al., 1973
; Greenwood and Titgemeyer, 2000). The catabolism of most AA is related to their supply in excess of those required for protein synthesis. In contrast, histidase activity, at least in rats, appears to be regulated by dietary protein intake and not by histidine supply (Kang-Lee and Harper, 1979; Torres et al., 1998, 1999). Hepatic histidase activity increases in response to an increase in protein intake. Feeding rats a diet high in His stimulates His oxidation without increasing histidase activity unless it is accompanied by a high-protein diet (Kang-Lee and Harper, 1977; Torres et al., 1998). Therefore, increasing protein intake when His is a limiting AA could increase the degradation of this essential AA and impede growth.
Ammonia (NH3) produced as a result of microbial fermentation in the rumen is absorbed through the ruminal wall and transported to the liver where it can be detoxified to urea. Studies in vivo (Lobley et al., 1995) and using hepatocytes in vitro (Mutsvangwa et al., 1997) have demonstrated that increasing NH3 leads to an increase in the oxidation of AA. This suggests that NH3 detoxification may contribute to the inefficient use of dietary AA, because as NH3 is detoxified to urea in the liver, AA can be consumed metabolically. However, other research suggests that AA use for the synthesis of urea from NH3 is not quantitatively important (Lobley et al., 1996; Luo et al., 1995; Milano et al., 2000).
Our objective was to determine the efficiency with which His is utilized by growing cattle and the effects of additional N supply in the form of NH3 and AA on whole-body protein deposition.
Materials and Methods
Experimental procedures for this study were approved by the Kansas State University Institutional Animal Care and Use Committee.
Experiment 1
Six ruminally cannulated Holstein steers (146 ± 7 kg; 161 ± 8 d of age) were housed in metabolism crates in a temperature controlled room (21°C) under continuous lighting. The experiment was of a 6 x 6 Latin square design. The treatments had a 3 x 2 factorial arrangement and included 0, 1.5, or 3 g/d L-His continuously infused into the abomasum and 0 or 80 g/d urea continuously infused into the rumen. Urea was used to increase the metabolic NH3 load. Experimental periods were 6 d long, which allowed 2 d for adaptation to treatment and 4 d of total fecal and urinary collection. Adaptation of 2 d is sufficient because ruminants rapidly adapt to changes in postruminal nutrient supply (Hovell et al., 1983; Moloney et al., 1998). Steers were adapted to the diet for 10 d before the start of the trial followed by a 4-d step-up period to adapt to infusions.
Steers had free access to water and were fed 2.5 kg/d (DM basis) of a diet based on soybean hulls (Table 1) twice daily in equal proportions at 0630 and 1830. The basal diet was formulated to provide low amounts of absorbable AA to the small intestine, and the metabolizable AA supply for this diet has been measured by Campbell et al. (1997). To ensure that His was the first-limiting AA for lean tissue deposition, a basal supply of 250 g/d of AA (Table 2) was infused abomasally (Greenwood and Titgemeyer, 2000). Because steers were limit-fed, additional energy was provided by infusing volatile fatty acids (VFA) into the rumen and glucose into the abomasum to ensure that energy supply was not as limiting as His supply. Infusions of VFA contained acetate (180 g/d), propionate (180 g/d), and butyrate (45 g/d) in a 4 kg/d solution. In addition, dextrose (300 g/d) was infused abomasally to provide additional energy without increasing microbial protein supply (Table 2). All steers received 10 mg/d pyridoxine•HCl, 10 mg/d folic acid, and 0.1 mg/d of cyanocobalamin to prevent them from limiting N retention (Lambert, 2001). All infusions were delivered via a peristaltic pump through polyvinylchloride tubing (0.24 cm i.d.) via the ruminal cannula. Abomasal infusions were delivered by inserting tubing through the reticulo-omasal orifice and the omasum into the abomasum, where it was held in place by a rubber flange (8-cm diameter).
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From d 3 through 6 of each period, feces and urine were collected daily; representative samples (10% of feces and 1% of urine) were saved, composited by steer within period, and frozen for later analysis. To prevent NH3 loss, urine was collected in buckets containing 300 mL of 6 M HCl. Feed samples and orts were collected, composited by period, dried at 55°C in a forced-air oven, and ground to pass a 1-mm screen. Feces, feed, and orts samples were analyzed for DM (105°C for 24 h) and OM (450°C for 8 h) to determine diet digestibility. Dry feed and orts and wet feces and urine samples were analyzed for N by total combustion (Nitrogen Analyzer Model FP-2000, Leco Corp., St. Joseph, MI) to determine N retention. Urine was analyzed for NH3 and urea concentrations colorimetrically (Technicon Industrial Systems, Buffalo Grove, IL; Method No. 337-74T and 339-01, respectively).
At the conclusion of the trial, steers were maintained on their respective treatments for an extra day to characterize ruminal NH3 concentrations. Ruminal fluid samples were taken from each steer 2, 4, and 6 h after feeding. To prevent NH3 loss, 0.5 mL of 6 M HCl was added to 10 mL of ruminal fluid that had been strained through four layers of cheesecloth. Samples were frozen and later thawed, centrifuged at 39,000 x g, and analyzed for NH3 as described for urine.
Blood samples were collected by jugular venipuncture into sodium heparinized vacuum tubes 5 h after the morning feeding on the last day of each period. Samples were immediately placed on ice and centrifuged for 20 min at 1,000 x g. Plasma was stored frozen and later analyzed for urea and glucose colorimetrically (Technicon Industrial Systems, Method No. 339-01 and SE4-0036FJ4, respectively). Plasma AA were measured using a commercial kit (EZ:faast; Phenomenex, Torrance, CA) to prepare AA for gas chromatography.
Statistical Analyses
. Data were analyzed using the MIXED procedure of SAS System for Windows Release 8.1 (SAS Inst. Inc., Cary, NC) with the fixed effects of steer, period, His, urea, and His x urea included in the model. Means were computed using the LSMEANS option. Linear and quadratic effects of His level and their interactions with urea were tested using contrasts for equally spaced treatments. None of the missing observations appeared to be treatment related.
Experiment 2
Six Holstein steers (150 ± 6 kg; 160 ± 7 d of age) fitted with ruminal cannulas were used in a 6 x 6 Latin square design with a 2 x 3 factorial arrangement of treatments. Experimental housing, periods, diet, basal infusions, sample collections, and laboratory analyses were the same as Exp. 1.
This experiment was designed to analyze the effect of supplemental AA on His utilization. Treatments were all supplied abomasally and included two levels of L-His (0 or 1 g/d), and three AA supplements, including no supplemental AA (Control), 100 g/d nonessential AA + 100 g/d essential AA (NEAA+EAA), and 200 g/d essential AA (EAA) delivered to the abomasum (Table 2
). Based on the results of Exp. 1, the level of 1 g/d supplemental His was considered to be less than the steers’ requirement for maximal N retention under the experimental conditions. The two groups of AA (EAA and NEAA+EAA) were evaluated in an effort to determine whether His metabolism was controlled more by the supply of EAA or of total AA-N to the steers. Because plasma concentrations of EAA are often more responsive to changes in dietary supply than those of nonessential AA, we hypothesized that the EAA treatment could alter His utilization to an extent greater that of than the NEAA+EAA treatment.
Abomasal infusates (Table 2) were prepared similarly to those in Exp. 1, with the EAA and NEAA+EAA treatments being added to the infusions by replacing 2 kg/d of water. For the EAA treatment, branched-chain AA (L-Leu, L-Ile, and L-Val) were dissolved in 1.76 kg of water containing 60.6 g of 6 M HCl. The remaining AA (L-Lys, L-Met, L-Arg, L-Thr, L-Phe, and L-Trp) were added to the solubilized mixture of branched-chain AA to bring the final weight of the mixture to approximately 2 kg. The NEAA+EAA mixture combined 1 kg of EAA solution (prepared as described above) and 1 kg of NEAA solution. The NEAA solution combined the Asp and Glu with 0.9 kg of water containing 11 g of NaOH. Once dissolved, the remaining NEAA (Gly, Ala, Pro, and Ser) were added to the mixture and dissolved.
The AA treatments were balanced for Na and Cl (resulting from the HCl and NaOH required to solubilize the AA treatments) by adding 32 g/d NaCl to the Control treatment, 22 g/d NaOH to the EAA treatment, and 16 g/d NaCl to the NEAA+EAA treatment.
Statistical Analyses
. Data were analyzed using the MIXED procedure of SAS with the fixed effects of steer, period, His level, AA treatment, and the His x AA interaction included in the model. Means for all treatments were computed using the LSMEANS option. Means for AA treatments and His x AA interactions were separated using F-test protected pairwise t-tests among all treatments. None of the missing observations appeared to be treatment related.
Results and Discussion
Experiment 1
Ruminal NH3 concentration was increased (P < 0.05) by the infusion of 80 g/d urea from 8.6 to 19.7 mM. Steers not receiving the urea infusion maintained ruminal NH3 concentrations adequate for normal ruminal fermentation, so differences in ruminal function would not be expected as a result of the urea treatment. Apparent total-tract DM and OM digestibilities (Table 3) were not different across treatment, averaging 77% and 79%, respectively, which also supports the concept that ruminal fermentation was not impacted by urea infusion.
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). Nitrogen intake increased by 36 g/d with the urea treatment (P < 0.01), whereas there were only small linear increases due to His infusion (P < 0.01). Fecal N was similar across treatment, suggesting that the infused treatment N was absorbed. Linear increases in N retention in response to His supplementation (P < 0.01) were linked to linear decreases in urinary N excretion (P < 0.01). Urinary urea N decreased (linear and quadratic, P < 0.05) with increasing His supplementation, whereas His did not affect urinary NH3 excretion.
Urinary N increased dramatically and in proportion to the increased N intake with the 80 g/d urea infusion (P < 0.01) such that N retention was unaffected by urea infusion. Urinary urea-N increased approximately 34 g/d and urinary NH3 increased by 1 g/d (P < 0.01) with the urea infusion (Table 3).
Although the quadratic response to His supplementation was not significant (P = 0.09), N retention responded to His, with the greatest increase between 0 and 1.5 g/d and a lesser response between 1.5 and 3 g/d. Because a near-maximal response was obtained with 1.5 g/d His, the requirement was probably met at a level only slightly above this point, with 3 g/d providing excess. The soybean hull-based diet (2.5 kg DM intake) provided 4.25 g/d His (Campbell et al., 1997), suggesting that the requirement for His was approximately 6 g/d for the maximal level of protein deposition that we observed. The incremental efficiency of His deposition—calculated over the response surface between 0 and 1.5 g/d supplemental His assuming that retained protein equals N retention x 6.25 and contains 2.5 g His/100 g of tissue protein (Ainslie et al., 1993)—averaged 65% (50 and 81% for no urea and 80 g/d urea treatments, respectively). Current protein models would predict the efficiency of AA use for the growth for our cattle (shrunk BW = 140 kg, frame score = 7) to be 71% (O’Connor et al., 1993; NRC, 1996), a value similar to what we observed. Infusion of urea did not significantly change N retention (P = 0.16), but both N retention and the efficiency of His utilization were numerically greater for steers receiving 80 g/d urea infusion.
Histidine is not usually considered to be the first-limiting AA in ruminant diets; however, previous research indicates that a deficiency in His can occur (Chalupa et al., 1973). Storm and Ørskov (1984) determined in sheep that ruminal microbes were deficient in His supply, but the His deficiency was small and both Met and Lys were more limiting than His. Greenwood and Titgemeyer (2000) determined that our soybean hull-based diet supplied deficient amounts of His, suggesting that our model would be useful for studying His utilization.
Ammonia produced within the rumen as a result of dietary protein fermentation is absorbed through the ruminal wall and transported to the liver for detoxification. Some evidence suggests that NH3 detoxification may contribute to the inefficient use of dietary AA because AA can be consumed metabolically during the process of urea synthesis (Lobley et al., 1995; Mutsvangwa et al., 1997, 1999). However, other research suggests that AA use for the synthesis of urea from NH3 is not quantitatively important (Luo et al., 1995; Lobley et al., 1996; Milano et al., 2000). Supplying 80 g/d urea to the rumen induced a metabolic NH3 load as indicated by the increased ruminal NH3 and plasma urea concentrations (2.7 mM for no urea vs. 5.1 mM for 80 g/d urea; Table 4) as well as urinary urea excreted. The N retention data indicated that the NH3 load did not have a negative impact on protein deposition, and, if anything, it was numerically increased by urea infusion. Subsequent research has found a similar lack of effect of an NH3 load on N retention when Met was the most-limiting AA (Awawdeh et al., 2003). Previous studies in sheep (Norton et al., 1982) and cattle (Moorby and Theobald, 1999) have also observed that an NH3 load did not impact N retention, but in those experiments the supply of protein or of a single AA was not limiting, so changes in protein deposition by the animal might not occur even if AA metabolism was affected by the NH3 load.
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). A numerically greater increase in plasma His was noted between the 1.5 and 3 g/d His than between 0 and 1.5 g/d His treatments, which reflects that the amount 3 g/d exceeded the steers requirement. No difference was noted in plasma His due to the urea infusion. Plasma Phe and Val decreased in response to supplemental His (P = 0.03), probably due to increased utilization for protein synthesis as the His deficiency was alleviated. Other essential AA also exhibited decreases in plasma concentrations as protein deposition increased, but these responses were not significant. Glutamine responded quadratically to His, with 1.5 g/d His leading to levels lower than those of either the 0 or 3 g/d amounts. Plasma Asp, on the other hand, tended to increase linearly (P = 0.06) in response to His supplementation. The magnitude of response in plasma Asp was not great, but large changes in neuroexcitatory AA levels, such as Asp, are unlikely to occur because their blood concentrations must be tightly controlled. Even when steers were abomasally infused with 80 g/d Asp, only small increases in plasma Asp concentrations were observed (Wessels and Titgemeyer, 1998). Circulating Gly concentrations decreased as a result of urea inclusion (P < 0.01), suggesting that Gly may have been removed by the liver as a source of non-NH3 N for urea synthesis. All of our steers received an abomasal supplement of 100 g/d Glu and 50 g/d of Gly as a source of nonessential AA, which increased the pool of N available for Asp synthesis, and, as a result, may have alleviated the need for essential AA to be used as a source of Asp-N in the process of detoxifying the NH3 load. Without this source of nonessential AA, it is possible that the NH3 load could have a negative impact on the usage of essential AA by the steers.
Ammonia escaping periportal hepatocyte ureagenesis stimulates glutamine synthetase in perivenous hepatocytes, producing Gln from Glu and NH3. Plasma Gln significantly increased (P < 0.01) in response to urea. This reflects the role of glutamine synthetase as a high-affinity scavenging system, which serves as a secondary route of NH3 detoxification (Haussinger et al., 1992). Plasma Glu numerically decreased in response to urea inclusion (P = 0.27). Although not considered a major route of NH3 detoxification under normal feeding situations (Parker et al., 1995), Glu production can serve a role in the removal of excess NH3 in the sheep liver (Wolff et al., 1972). Net changes in plasma Glu concentrations were not very dramatic, likely a result of increased production by glutamate dehydrogenase and increased utilization for Gln synthesis.
Experiment 2
No significant interactions were observed between His and AA treatments for diet digestibilities or for N retention data (Table 5). Diet DM and OM digestibilities were not altered as a result of treatment, averaging 72% and 74%, respectively. In contrast to Exp. 1, fecal N (Table 5) decreased (P < 0.01) in response to His inclusion. It is unknown whether this response was related to a change in intestinal digestion or to a change in endogenous losses into the gut; regardless, decreases in fecal N are not typically observed in response to supplementation with a limiting AA, so this response was unexpected. Although His did not significantly affect total urinary N (Table 5) or its components (Table 5), N retention was increased (P = 0.02) as a result of His supplementation. Although fecal N was the only component used in the calculation of N retention that statistically responded to His supplementation, the majority of the N retention response cannot be attributed to it. About one-third (1.3 g/d) of the 4.1 g/d increase in N retention in response to His supplementation was attributable to the observed decrease in fecal N, whereas the remaining two-thirds were attributable to the nonsignificant decrease in urinary N excretion (1.7 g/d) and the slightly higher N intakes (1.1 g/d). The responses in N retention to His were numerically greater for the NEAA+EAA and EAA treatments than for Control (i.e., increases of N retention in response to His of 3.6 and 6.3 g/d vs. 2.3 g/d). Because N retention was increased in Exp. 1 when steers were provided with more than 1.5 g/d supplemental His, the level of His supplementation in Exp. 2 (1 g/d) was selected to ensure that it would fall within the linear response surface. Consequently, His efficiency can be determined between 0 and 1 g/d supplemental His. In agreement with Exp. 1, incremental His efficiency averaged 63% over the AA treatments (36%, 56%, and 98% for Control, NEAA+EAA, and EAA, respectively).
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, 1979; Tanaka and Ogura, 1980; Torres et al., 1998, 1999; Tovar et al., 2002). Our trial increased absorbable N supply from 73.4 g/d for Controls to 99.2 g/d for AA treatments, which might not have increased AA supply enough to stimulate histidase activity sufficiently to increase His catabolism. It is also possible that the regulation of histidase activity in cattle differs from that in rats.
The addition of NEAA+EAA increased (P < 0.05) N intake as well as urinary N excretion (Table 5). The amount of NH3-N found in the urine was greatest (P < 0.05) for the NEAA+EAA and No His treatment. Urinary urea levels were greater (P < 0.05) for NEAA+EAA than for Control. Nitrogen retention was increased as a result of the NEAA+EAA treatment because increases in urinary N were less than increases in N intake. Treatments were not balanced for energy. The additional AA provided by the NEAA+EAA treatment were used for deposition with an average efficiency of only 17%, suggesting the AA were used as an energy source because the AA treatments were provided in excess of the requirement for all AA except His. Protein deposition is linked to energy availability, and the use of AA as an energy source to drive protein deposition leads to efficiencies similar to our observed value (Asplund, 1994). In response to the additional energy supply, steers receiving the NEAA+EAA treatment had greater protein deposition than Control steers (P < 0.05) even when His was deficient.
Similar to the NEAA+EAA treatment, the EAA treatment increased (P < 0.05) N intake by 26.3 g/d (Table 5). Urinary N excretion was increased (P < 0.05) by EAA when compared with Control, predominantly as a result of increased urinary urea excretion (Table 5). In contrast to the NEAA+EAA treatment, steers on the EAA treatment did not exhibit improved N retentions. Both treatments provided 200 g/d of additional AA that could be used as an energy source. However, we believe the EAA treatment induced a Met imbalance, which may have moderated responses to the energy provided by the EAA treatment. Abe et al. (1999) reported that growing calves given 34.2 g/d DL-Met had significantly lower N retentions as a result of a Met imbalance, whereas calves given 20.7 g/d of DL-Met did not exhibit a reduction in N balance. We provided 30 g/d of supplemental L-Met with the EAA treatment but only 20 g/d with the NEAA+EAA treatment. Plasma Met concentrations (Table 6) were extremely high for the EAA treatment, which reflects the Met imbalance. In contrast, plasma Met concentrations were not greatly increased by the NEAA+EAA treatment. Plasma Met levels of steers given 34.2 g/d DL-Met and exhibiting signs of an imbalance averaged 1,894 µM (Abe et al., 1999), which is greater than we observed (993 µM), but differences could in part be due to the fact that Abe et al. (1999) utilized DL-Met whereas we used L-Met. The numerically greater efficiency of His utilization observed for EAA than for Control and NEAA+EAA may be because the Met imbalance was lessened in response to an increased His supply.
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) was reduced in response to His (P < 0.01) but was unaffected by the NEAA+EAA and EAA treatments. Plasma His concentrations (Table 6) were greater when His was supplemented, but the inclusion of His did not alter concentrations of other plasma AA. Histidine concentration was lower for both AA treatments when compared to the Control (P < 0.05), possibly indicating that additional AA available as an energy source increased protein synthesis and consequently utilized more His. However, His degradation via histidase is regulated by dietary protein in rats. Increasing dietary protein intake increased histidase activity (Kang-Lee and Harper, 1979; Torres et al., 1998, 1999), whereas an increase in His intake had no effect on histidase activity in rats (Kang-Lee and Harper, 1977; Torres et al., 1998). This suggests that histidase activity is regulated by total dietary protein instead of His. Hence, lower plasma His concentrations for the steers receiving NEAA+EAA or EAA could be indicative of increased histidase activity. In contrast to histidase activity, His oxidation is typically related to its supply (Kang-Lee and Harper, 1977, 1979), consequently, when His is limiting, it is spared from oxidation and used more efficiently for protein synthesis. Histidine oxidation may not have been increased in those steers given the NEAA+EAA and EAA treatments because increased protein deposition competed with histidase for His. The increased protein deposition may have decreased His concentrations and subsequently spared His from oxidation. Greater increases in N retention in response to supplemental His when NEAA+EAA or EAA were provided suggest that His oxidation was decreased by the treatments. The NEAA+EAA and EAA treatments significantly affected plasma concentrations of all AA with the exception of Asn. The nonessential AA Ala, Pro, and Ser increased (P < 0.05) with the NEAA+EAA treatment, likely as a direct result of the increased supply. Likewise, the essential AA Ile, Leu, Lys, Phe, and Val increased (P < 0.05) with infusion of the NEAA+EAA treatment. Methionine concentrations numerically increased with NEAA+EAA, but the increase was not significant due to the high variation induced by the Met imbalance for the EAA treatment.
Infusion of the EAA treatment, compared with Control, increased (P < 0.05) plasma levels of Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val, likely because of their increased supply. Tyrosine levels for the EAA treatment were increased (P < 0.05), likely due to the increased supply of Phe, its precursor. Likewise, when compared with the Control, 
-aminobutyric acid, which can be produced from a transsulfuration product of Met (-ketobutyric acid), likely increased (P < 0.05) because of an increased supply of its precursor AA from the EAA treatment. Plasma Pro concentrations were also greater for EAA than control, although this AA was not provided by that treatment.
The EAA treatment also increased plasma concentrations of Lys, Met, Phe, Thr, and Val above that observed for the NEAA+EAA treatment, likely on account of the greater amount infused; a similar response for Tyr likely resulted from the greater amount of Phe infused. There was significantly more -aminobutyric acid for the EAA treatment compared with the NEAA+EAA treatment, reflective of the high levels of circulating Met obtained from the EAA infusion. In agreement with the findings of Lobley et al. (2001), who infused high levels of AA, ornithine increased (P < 0.05) with AA treatments compared to Control.
Aspartic acid, Glu, Gln, and Gly all exhibited lower plasma concentrations as a result of EAA when compared to the Control and NEAA+EAA treatments, presumably in response to urea production. Previous research on infusing an excess supply of AA into the mesenteric vein of cattle and sheep has found an increase in urea production in response to an increased AA supply (Wray-Cahen et al., 1997; Lobley et al., 1998). High levels of urinary and plasma urea concentrations suggest that the AA treatments exceeded the steers’ requirement for protein deposition and were subsequently degraded and removed from the system via ureagenesis. The lower levels of circulating Gly for the EAA treatment support findings in Exp. 1. This implies that Gly served as a non-NH3 source of N to aid in the detoxification of NH3. Another possible use for Gly in steers infused with EAA would be for Ser synthesis to increase Met disposal via transsulfuration.
Aspartic acid followed the same trend as Gly, suggesting that it also was used in the process of urea synthesis. Aspartate, which donates a N for the synthesis of urea, is thought to be derived from Glu through transamination reactions and would, therefore, require an input of AA as its immediate source of N (Parker et al., 1995). Plasma concentrations of Gln and Glu were numerically (NEAA+EAA) and significantly (EAA) lower than Control. Increased removal of Gln by the liver occurs with increasing levels of AA infusion (Wray-Cahen et al., 1997). The extracted Gln is acted upon by glutaminase to release NH3, which is then routed to carbamoyl phosphate synthesis due to its close spatial relation to glutaminase in the mitochondria (Meijer et al., 1990). When excess AA are absorbed, the amino-N produced can be transferred, during degradation of excess AA, to Asp via Glu for urea synthesis. Consequently, Gln plays a vital role in urea production by balancing N inputs to the urea cycle through carbamoyl phosphate (Lobley and Milano, 1997), whereas Glu is utilized for urea synthesis via transamination to Asp.
General Discussion
Hepatic ureagenesis serves three main purposes. It prevents toxic NH3 from reaching peripheral tissues, disposes of AA-N surplus to anabolic requirements, and aids in regulation of acid-base balance. Elevated plasma and urine urea concentrations in response to ruminal urea infusions in Exp. 1 indicate that the resultant NH3 load was predominantly detoxified to urea. Likewise, the increases in urea excretion in response to AA supplementation in Exp. 2 suggest that the excess AA were also converted predominantly to urea. Previous research studying the effects of NH3 on metabolism has indicated that, with administration of NH4Cl, mild acidosis can initiate protein breakdown and AA oxidation (Reaich et al., 1992; Lobley et al., 1995). Low amounts of NH3 in the urine verify, in both of our experiments, that steers were not experiencing any significant metabolic acidosis. Therefore, our treatment responses do not appear to be confounded with a metabolic acidosis.
Ainslie et al. (1993) developed an equation to predict the efficiency of absorbed protein utilization for growth based on body weight: 0.834 - 0.00114 x equivalent shrunk weight. This equation suggests that the efficiency of AA use for growth decreases as cattle increase in body weight, but that all AA are used at the same efficiency. Based on this equation, the predicted efficiency by which AA were used for growth under our experimental conditions was 71%, which is near our measured efficiencies of 65 and 63% for Exp. 1 and 2, respectively. In Exp. 1, the efficiency of His use between 1.5 and 3 g/d was only 13%, suggesting the requirement was probably met below 3 g/d His. Heger and Frydrych (1989) found maximum AA efficiencies when the AA was provided within 30 to 60% of the optimal requirement, but above that range AA efficiency decreased.
Our estimates of the efficiency of His utilization are based on the average His content of tissue proteins as reported by Ainslie et al. (1993). However, the studies determining empty-body protein composition report His content to range from 1.3 to 3.3 g His/100 g tissue protein, with Ainslie et al. (1993) reporting a value of 2.1 g His/100 g tissue protein from their own research. The average value was 2.5 g His/100 g tissue protein, which is also the value adopted by the NRC (1996). If the actual amount of His in tissue protein is not 2.5%, then our estimates of the efficiency by which His is utilized will be biased because we based our efficiency estimates on the amount of His deposited, which was calculated with the assumption of 2.5 g His/100 g tissue protein.
In Exp. 1, when N retention was maximized (N balance = 39.0 g/d), deposition of His was predicted to be 6.1 g/d, which is near the observed requirement based on N retention responses. Clearly, efficiencies of use near 65% would necessitate that the requirement for growth exceed the amount deposited by about one-half. Maintenance requirements, estimated by equations of O’Connor et al. (1993) to be around 2.1 g/d His, would also contribute to an even greater need for His. Thus, it is clear that the estimated maximal deposition of 6.1 g/d His and a requirement of around 6 g/d His cannot be simultaneously correct. Much of the discrepancy between the observed His requirement near 6 g/d and the predicted value of 10.7 g/d (growth needs of 8.6 g/d [6.1/0.71] plus maintenance needs of 2.1 g/d) can be explained by the fact that N retention typically overestimates protein deposition. If protein deposition was lower than predicted by N retention, then the predicted amount of His deposited and, subsequently, the His requirement would both be lower. However, it is also possible that the efficiency of His utilization below the 4.25 g/d His provided by the diet could be greater than that observed between 0 and 1.5 g/d supplemental His, and this also would contribute to a lower His requirement. As discussed previously, our estimates of the efficiency of His utilization are dependent upon the predicted His content of protein gain. However, any misestimation of efficiency for this reason will not impact the predicted requirement for His when protein deposition is the response criterion (i.e., His requirement for growth = His deposition/efficiency of utilization) because misestimates in His deposition rates counteract misestimates in the efficiency of His utilization.
In both of our experiments, we observed numerical differences in the efficiency of His utilization in response to supplementation with nitrogenous compounds (ammonia or AA load). This suggests that nutritional factors do impact how efficiently AA (at least His) are used, but further work will be needed before these variables can be incorporated into protein models used for predicting animal performance.
Implications
Supplying histidine to diets deficient in metabolizable histidine supply can improve protein deposition by growing cattle. Determining nutritional factors that influence the efficiency with which histidine is utilized in cattle can improve current methods of balancing diets to meet protein requirements. Increases in ammonia and amino acid loads did not demonstrate a metabolic cost in terms of whole-body protein deposition, regardless of whether histidine was limiting. Consequently, although an excess protein supply may not be economically efficient or environmentally friendly, it does not seem to directly penalize animal performance.
Footnotes
1 Contribution No. 04-034-J from the Kansas Agric. Exp. Stn., Manhattan. 
2 Correspondence: 132 Call Hall (e-mail: etitgeme{at}oznet.ksu.edu).
Received for publication August 22, 2003. Accepted for publication November 6, 2003.
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