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ANIMAL NUTRITION |
* Department of Animal Sciences and Industry and
Department of Clinical Sciences, Kansas State University, Manhattan 66506-1600
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: cattle • energy • growth • leucine • utilization
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Chiba et al., 1991). This type of relationship between the effects of energy and protein supplies on protein deposition has been described as the protein-and energy-dependent phases of growth (Gerrits et al., 1996; Titgemeyer, 2003), and it demonstrates that the efficiency of AA use is not affected by the level of energy intake. Although information on growing ruminants is limited, nutrient requirement systems have assumed a constant efficiency of AA utilization for growing cattle (Ainslie et al., 1993).
In previous studies with growing steers, we observed that, even when Met limited protein accretion, N retention increased in response to increased energy supplementation, independent of the source of supplemental energy (Schroeder et al., 2006a,b). We concluded that the assumption of a constant efficiency of AA utilization by growing cattle is unlikely to be appropriate, and that energy level should be considered to more precisely estimate AA requirements.
It is unknown whether the observed positive effects of energy supply on Met utilization are of similar magnitude for other AA, which are metabolized differently. For instance, Met is primarily catabolized by trans-methylation followed by transsulfuration, whereas Leu is catabolized throughout the body by transamination as the initial step. Because of these differences, regulation of their metabolism may be different, and energy supply could have different effects on their utilization. Moreover, whether BW (stage of maturity) affects AA utilization in growing steers is not known.
Our objective was to determine the effects of energy supplementation on Leu utilization by growing steers at 2 initial BW.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Procedures were approved by the Kansas State University Institutional Animal Care and Use Committee.
Two experiments were carried out with the same 6 ruminally cannulated Holstein steers. In Exp. 1, the steers (150 ± 7 kg of initial BW; 180 ± 7 d of age) were allocated in a 6 x 6, balanced, Latin square design. The steers were housed in individual metabolism crates with continuous lighting and controlled temperature (22°C). The animals had continuous access to fresh water and were limit-fed, at 2.3 kg of DM/d (48.3 g of OM/ kg of BW0.75 daily), a diet based on soybean hulls (Table 1) in 2 equal portions at 12-h intervals. Diet composition (Table 1) and feed restriction were designed to provide the estimated maintenance energy requirements (6.0 Mcal of ME/d) and to supply small amounts of dietary AA to create a limitation in Leu.
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). Amino acid solutions for each steer were prepared by dissolving the Ile and Val in 1.0 kg of water containing 40 g of 6 M HCl. Once these AA 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 2 solutions were mixed together, and Leu or 6 M HCl (see below) and water were then added to bring the total weight of the daily infusate to 4 kg.
All steers received 10 mg of pyridoxine·HCl/d, 10 mg of folic acid/d, and 100 µg of cyanocobalamin/d mixed with the abomasal infusate, to avoid deficiencies in those vitamins (Lambert et al., 2004). In addition, all steers received a basal infusion of 100 g of acetate/d, 75 g of propionate/d, and 75 g of butyrate/d into the rumen and 200 g of glucose/d into the abomasum as energy sources to provide an additional 1.9 Mcal of GE/ d without affecting microbial protein synthesis. The VFA were infused as free acids and mixed with water, such that the total weight of the ruminal infusate was 4 kg/d, and the glucose was mixed with the basal AA infusate.
Treatments were arranged in a 3 x 2 factorial, with abomasal infusion of 3 amounts of L-Leu (0, 4, or 8 g/ d) and supplementation with 2 amounts of energy (0 or 1.9 Mcal of GE/d). Leucine was dissolved in water containing 20 g of 6 M HCl and was added to the basal AA mixture according to treatment. Additional solutions of 20 g of 6 M HCl and water were prepared to equalize the acid supply. The amounts of supplemental Leu were based on a previous study with the same experimental model (Awawdeh et al., 2005), which demonstrated that supplemental Leu requirements are close to 8 g/d. Energy supplementation (1.9 Mcal of GE/ d) was within a linear range of responses observed in previous studies that used Met as the limiting AA (Schroeder et al., 2006a), and was achieved by continuously infusing additional amounts of VFA (100 g of acetate/d, 75 g of propionate/d, and 75 g of butyrate/d) into the rumen and additional glucose (200 g/d) into the abomasum. Therefore, steers receiving the energy supplementation treatment received a total energy infusion of 3.8 Mcal of GE/d (1.9 Mcal/d from the basal infusion plus 1.9 Mcal/d from the treatment), whereas control steers received only 1.9 Mcal/d from the basal infusion. The supplemental energy sources were added to the basal infusions, such that the total weight of ruminal and abomasal infusates was 4 kg/d for all treatments. A peristaltic pump and polyvinylchloride tubing (2.4 mm i.d. Tygon R-3603, Saint-Gobain Performance Plastics, Akron, OH) were used to infuse solutions into the rumen and abomasum. Abomasal lines were placed through the reticulo-omasal orifice and into the abomasum, and were retained by a rubber flange (8-cm diameter) attached at the end of the line.
Sample Collection and Analyses
Each experimental period consisted of 2 d for adaptation and 4 d for sample collection. It was previously demonstrated in our experimental model (Schroeder et al., 2006b) that cattle adapt (i.e., reach steady-state N balance) within the 2-d time frame to changes in nutrient supply when both protein and energy supplies are varied and ruminal adaptation is not required. Feed samples were collected from d 2 through 5 of each period, composited by period, and stored (–20°C) for later analysis. Steers typically consumed their feed allotments within 6 h; however, feed refusals (if any) were fed through the ruminal cannula (up to 1.2 kg of DM/ d) approximately 1 h before the next feeding. Total urinary (collected into buckets containing 1.3 L of 1.38 M HCl to keep the pH below 3) and fecal outputs were collected daily, with samples of urine (1%) and feces (10%) saved, composited by period within animal, and stored at –20°C. Before analysis, samples were thawed at room temperature and homogenized. Feed and fecal samples were partly dried at 55°C for 36 h, air-equilibrated for 36 h, and ground with a Wiley mill (Thomas Scientific, Swedesboro, NJ) to pass a 1-mm screen. Partly dried diet and fecal samples were analyzed for DM (105°C for 24 h) and ash (450°C for 8 h). Total N was determined for the diet, wet fecal samples, and urinary samples with a Leco FP 2000 N Analyzer (Leco, St. Joseph, MI). Urine samples were analyzed colori-metrically for NH3 (Broderick and Kang, 1980) and urea concentrations (Marsh et al., 1965).
On d 6 of each period, 4 h after the morning feeding, jugular blood was collected into vacuum tubes containing sodium heparin (Becton Dickinson, Franklin Lakes, NJ). Blood samples were immediately placed on ice and then centrifuged for 20 min at 1,000 x g to obtain plasma, which was frozen (–20°C) for later analysis. Plasma was analyzed for glucose and urea as described by Schroeder et al. (2006a). Blood samples were also collected in tubes without anticoagulant, left for 30 min at room temperature, and centrifuged for 20 min at 1,000 x g, and the serum was stored (–20°C) for later analysis of insulin by an RIA kit (DSL-1600, Diagnostic Systems Laboratories Inc., Webster, TX; intraassay CV of 9.1% and a sensitivity of 0.043 ng/mL).
Statistical Analyses
One animal did not satisfactorily tolerate the energy supplementation, as demonstrated by refusals of large amounts of feed, and all data from 2 periods were not used for analyses. For the third assignment of energy supplementation to this steer, the energy supplementation was not provided, which yielded an additional observation for the treatment with no supplemental energy and no supplemental Leu. All data from 1 other observation (energy supplementation, no Leu) were lost because of problems not related to treatment. Statistical analyses were performed by using PROC MIXED (SAS Inst. Inc., Cary, NC). The model included the effects of Leu, energy, Leu x energy, and period. Steer was included as a random variable. Linear and quadratic effects of Leu supplementation and their interactions with energy were tested by using single df orthogonal contrasts. Treatment means were determined by using the LSMEANS option.
When Exp. 1 was finished, steers were housed in an outdoor pen for 89 d and fed a diet based on corn silage for ad libitum consumption. Once the steers reached the target BW (275 ± 12 kg, 312 ± 7 d), Exp. 2 was conducted, with housing, periods, diet (Table 1
), basal infusions, treatments, sample collection, laboratory analysis, and statistical analysis similar to Exp. 1. The only adjustment was that feed intake was 3.6 kg of DM/ d to provide a similar amount of feed on a metabolic BW basis (48.6 g of OM/kg of BW0.75 daily) and supply the estimated energy maintenance requirements (9.8 Mcal of ME/d). In Exp. 2, all data from 1 observation were lost because of problems unrelated to treatment.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Nitrogen balance and diet apparent total tract digestibilities for Exp. 1 are presented in Table 2. The infused nutrients (VFA, glucose, and AA) were assumed to be totally digestible and were not accounted for in the calculations of digestibilities. Apparent digestibilities of DM and OM were similar among treatments, and averaged 71.0 and 75.4%. Total N intake was not affected by energy supplementation, but was linearly increased by Leu (P < 0.05) because of the N provided by the Leu. Fecal N excretion was similar among treatments. Leucine supplementation linearly decreased (P < 0.05) excretion of total and urea N in urine. In addition, the interaction of the linear effect of Leu x energy was significant (P < 0.05), indicating that energy supplementation decreased urinary N excretion when 4 or 8 g/d of Leu was supplied, but not when Leu was not supplemented. The interaction of the linear effect of Leu x energy tended to be significant (P = 0.06) for N retention, because the response to Leu supplementation was dependent on energy supplementation. When supplemental energy was not provided, N retention was increased by increasing Leu supplementation from 0 to 4 g/d, but there were no further changes by increasing Leu supplementation to 8 g/d. When the steers received additional energy, there was a linear increase in N retention in response to Leu supplementation.
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) presented a pattern similar to that observed for urinary urea N excretion. Energy supplementation increased (P < 0.05) plasma glucose (6.1 vs. 5.6 mM) and serum insulin concentrations (1.02 vs. 0.58 ng/mL), but glucose and insulin were not affected by Leu supplementation. Exp. 2
Nitrogen balance and diet apparent total tract digestibilities for Exp. 2 are presented in Table 3. Apparent DM and OM digestibilities were linearly increased by Leu supplementation (P < 0.05). In addition, apparent OM digestibility was negatively affected by additional energy supply (76.2 vs. 74.4%; P < 0.05). Changes in apparent OM digestion matched with the observed fecal N excretion, which was greater when steers received supplemental energy at the lowest levels of Leu supplementation (0 and 4 g/d), but was less in response to energy supplementation at the highest level of Leu (Leu x energy interaction, P < 0.05). The experimental design led to linear increases in total N intake as the amount of Leu infusion increased, although the magnitude of that increase was small (<1 g of total N intake/d). Total and urea N excretions in urine were significantly reduced by additional energy supply, but they were not affected by Leu supplementation (Table 3). Retention of N was not affected by Leu supplementation, but was increased (P < 0.05) by energy supplementation (33.0 vs. 27.8 g/d) because of the reductions (P < 0.05) in urinary N excretion (31.3 vs. 37.9 g/d).
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) were lower (3.0 vs. 2.4 mM; P < 0.05) when additional energy was supplied, with no effect of Leu supplementation. As observed in Exp. 1, energy supplementation was associated with significant increases in plasma glucose (5.4 vs. 5.1 mM) and serum insulin concentrations (0.94 vs. 0.62 ng/mL). |
DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Diet digestibility was not affected by treatment in Exp. 1 (Table 2), but it was linearly increased by Leu supplementation in Exp. 2 (Table 3). Although changes in apparent OM digestion are not typically observed by infusion of the most limiting AA, small increases have also been observed when supplemental Leu (Awawdeh et al., 2005) or Met (Schroeder et al., 2006a) was provided to growing steers. Nevertheless, in our study, the changes were not of a large enough magnitude to alter the interpretation of our results. The moderate reduction in apparent OM digestion caused by energy supplementation in Exp. 2 (Table 3) was also observed in previous studies when additional VFA (primarily acetate) were infused into the rumen (Schroeder et al., 2006a,b). Because total VFA infusion was similar in both experiments, we cannot explain why apparent OM digestion was affected only in Exp. 2, in which total DMI was greater and the amounts of VFA infused were less if expressed on a metabolic BW basis. Although it is possible that the supply of Leu would be reduced by the energy supplementation in Exp. 2, total Leu supply was not limiting in Exp. 2 (see discussion below); therefore, it does not affect the interpretations regarding responses to Leu and energy supplies.
In Exp. 1, when additional energy was not provided, N retention was increased by increasing Leu supplementation from 0 to 4 g/d, with no additional increases when supplemental Leu was increased from 4 to 8 g/d (Table 2). We interpreted these results to indicate that the supplemental Leu requirement was, at most, not much greater than 4 g/d. The potential for protein deposition in growing animals has been described as an energy-driven process (Gerrits et al., 1996; Titgemeyer, 2003), being greater with increased energy supply. Therefore, when steers receive additional energy supply, the potential for protein deposition might be greater, increasing the ability of the steers to respond to higher levels of supplemental Leu supply, and thus the Leu requirements. When steers were provided with an additional 1.9 Mcal of GE/d, the supplemental Leu requirement was greater than 4 g/d and, based on linear increases in N retention, was at least 8 g/d. Changes in plasma urea concentration were in accordance with those observed in urinary N excretion (Table 2), decreasing in response to Leu supplementation, with the decrease being greater when additional energy was supplied. In agreement with our results, Awawdeh et al. (2005), using a similar diet and intake level (48 g of OM/kg of BW0.75 daily) and a basal energy supplementation of 3.1 Mcal of GE/d (intermediate value between the 1.9 and 3.8 Mcal of supplemental GE/d in our study), observed that supplemental Leu requirements were greater than 4 g/d, and likely close to 8 g/d. If retained N was deposited as tissue protein (N x 6.25) and tissue protein gain contained 6.7% Leu (Ainslie et al., 1993), the estimated incremental efficiency of supplemental Leu use was 
26% for control steers and 30% for energy-supplemented steers at 0 to 4 g of supplemental Leu/d [(27.0 – 24.5) x 6.25 x 0.067/4 for control steers; (28.5 – 25.6) x 6.25 x 0.067/4 for energy-supplemented steers]. These values represent the incremental efficiency in the utilization of Leu under conditions in which Leu is clearly the most limiting nutrient. Those values were slightly less than those (34 to 49%) observed in previous studies (Awawdeh et al., 2005, 2006) and much less than the value predicted (66%) by the NRC (1996). Energy supplementation increased (P < 0.05) N retention at the same amounts of Leu supply (Table 2), although the responses in the presence of 0 and 4 g/d of Leu would not be statistically significant if tested with pairwise comparisons. By dividing the total Leu retention (N retention x 6.25 x 0.067) by the Leu supply from the diet (5.9 g of metabolizable Leu/kg of DMI; Campbell et al., 1997), we calculated that the gross estimated efficiency of use of dietary Leu was numerically increased from 75 to 79% by energy supplementation when supplemental Leu was not provided. At 4 g/d of Leu supplementation, the estimated gross efficiency of Leu utilization increased numerically from 64 to 68% because of additional energy supply. Although the absolute values for these gross efficiencies are likely overestimated by overestimations inherent within the N retention procedure (Gerrits et al., 1996), the relative changes suggest that the additional energy supply improved the efficiency of Leu utilization. In previous studies, we observed that, when Met limited protein deposition, energy supplementation increased the efficiency of Met utilization (Schroeder et al., 2006a,b), although the magnitude of the improvement in efficiency of use seems to be greater for Met than for Leu. Differences in the metabolism of these 2 AA, Met primarily catabolized via transmethylation followed by transsulfuration and Leu catabolized throughout the body via transamination, may partly explain the differences in the magnitude of the effects of additional energy supply. However, more research is needed to identify the specific tissues and mechanisms involved in changing the efficiency of AA utilization by energy supplementation.
In Exp. 2, when the experiment was repeated with the same steers, but weighing 275 kg of BW, the lack of response in N retention to Leu supplementation (Table 3) indicated that Leu did not limit protein deposition. Although there was no previous information on Leu requirements for steers of that BW, the lack of response was unexpected. According to the equations used by the Cornell Net Carbohydrate and Protein System for estimating Leu requirements (O’Connor et al., 1993), 2 of the 3 determinants of maintenance requirements increase with BW, although with an exponent smaller than 0.75 (0.6 for scurf protein and 0.5 for urinary protein). By maintaining a similar DMI between Exp. 1 and Exp. 2 on a metabolic BW basis (using the exponent 0.75), we provided more Leu (g/d) in Exp. 2, and the increase in supply was slightly greater than the increase in maintenance Leu requirements. Basal supply of Leu was 161 and 195% of the estimated (O’Connor et al., 1993) maintenance requirements in Exp. 1 and Exp. 2, respectively. Comparing the estimated maintenance Leu requirements (8.4 and 12.7 g/ d for Exp. 1 and 2; O’Connor et al., 1993) with the basal supply of 5.9 g of metabolizable Leu/kg of DMI (Campbell et al., 1997), Leu supply available for growth was 5.2 and 12.1 g of metabolizable Leu/d in Exp. 1 and Exp. 2, respectively. Based on the equation generated by Institut National de la Recherche Agronomique (1989) [efficiency = 0.834 – 0.0114 x equivalent shrunk BW (kg)] and adopted by several nutrient requirement models (Ainslie et al., 1993; O’Connor et al., 1993; NRC, 1996), the incremental efficiency of absorbed Leu utilization for growth was predicted to decrease from 66 to 52% from Exp. 1 to Exp. 2. Although a lower incremental efficiency for growth in Exp. 2 than in Exp. 1 could partly compensate for the greater basal Leu supply, the Leu available for growth was 2.3-fold greater in Exp. 2, allowing greater N retention and limiting the capacity of steers to respond to supplemental Leu. Regardless, results indicated that the steers were in an energy-dependent phase of growth during Exp. 2, with the potential for increased protein deposition in response to additional energy (Table 3). Gross efficiency of Leu utilization was clearly increased by energy supplementation in Exp. 2, but this simply reflects a greater capacity of the steers for protein deposition and, because Leu was not a limiting nutrient, provides no information regarding the true efficiency of Leu utilization.
In our experimental model, in which severe deficiencies in glucose are unlikely, additional glucose and pro-pionate supplies were previously associated with increased plasma glucose concentrations (Schroeder et al., 2006b). In agreement, significant increases in plasma glucose and serum insulin concentrations (Tables 2 and 3) were observed in both Exp. 1 and Exp. 2, when steers received additional VFA and glucose as supplemental energy. Insulin may play an important role in mediating the nutritional effects on muscle protein deposition, which affects the efficiency of AA utilization in growing animals (Davis et al., 2003). Although positive relationships between plasma insulin concentrations and increases in AA utilization have been observed (Rooyackers and Nair, 1997), we also found that increasing energy supply through lipogenic energy sources increased N retention without affecting serum insulin concentrations (Schroeder et al., 2006b), suggesting that insulin may not be the most critical regulator of protein deposition in our model. In a previous study with growing steers (Awawdeh et al., 2006), we observed that when Leu was the most limiting AA, Leu supplementation increased serum insulin but did not affect plasma glucose concentrations. In the current study, Leu supplementation did not affect plasma glucose or serum insulin concentrations (Tables 2 and 3).
The current study, in conjunction with our previous studies, indicates that the assumption of a constant efficiency of AA utilization for all the essential AA and across different levels of energy supply may not be appropriate for estimating AA requirements of growing steers. Modeling of AA requirements in growing cattle may require consideration of the amount of dietary energy supplied. Moreover, the magnitude of the effect of energy supplementation on the efficiency of AA use may be different, depending on which AA limits protein deposition.
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Footnotes |
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2 Corresponding author: etitgeme{at}ksu.edu
Received for publication December 1, 2006. Accepted for publication June 27, 2007.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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