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ANIMAL NUTRITION |
* Department of Animal Sciences and Industry, and
and
Department of Clinical Sciences, Kansas State University, Manhattan 66506-1600
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONIMPLICATIONSLITERATURE CITED |
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Key Words: methionine • energy • growth • utilization
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONIMPLICATIONSLITERATURE CITED |
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found in pigs that when energy was limiting, protein deposition did not change in response to protein supply. When energy was adequate, protein deposition in pigs increased linearly with Lys supply, but when Lys limited protein retention, increased energy did not affect protein deposition (Chiba et al., 1991). The relationship between energy and protein supplies on protein deposition in pigs has been described as protein-and energy-dependent phases of growth (Titgemeyer, 2003). With this model, the efficiency of AA use is not affected by energy intake, and AA requirements may be expressed relative to energy intake.
Protein- and energy-dependent phases of growth have been assumed for growing cattle by most nutrient requirements systems (Ainslie et al., 1993). Because of the difficulty in varying energy intake without affecting ruminal microbial protein production, the relationship between AA and energy supplies on protein deposition is poorly understood in ruminants. Lindberg and Jacobsson (1990), using sheep intragastrically infused with increasing protein at 3 rates of energy supplementation, found a linear relationship between N infusion and N retention when energy supply was high. At lower energy supplementation rates, N retention increased initially, then plateaued as N supply increased, indicating protein- and energy-dependent phases of growth. In preruminant calves, increasing energy intakes improved protein retention at both high and low protein intakes (Gerrits et al., 1996), suggesting that energy intake affects efficiency of AA use, challenging the assumption of a constant efficiency of AA use across different energy levels.
Our objective was to determine effects of energy supply on Met use for whole-body protein retention in growing steers. We hypothesized that increased energy supply would improve efficiency of Met use.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONIMPLICATIONSLITERATURE CITED |
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).
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 (2.82 kg of DM/d) a diet based on soybean hulls (Table 1) at 12-h intervals. The basal diet provided a low protein:energy ratio, small amounts of ruminally undegradable protein, and enough ruminally available N to maximize ruminal microbial growth (Campbell et al., 1997). Feed restriction was designed to maintain a small supply of AA to create a limitation in Met, such that a clear response to its supplementation could be achieved.
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). Amino acid solutions for each steer were prepared by dissolving L-Leu, L-Ile, and L-Val in 1 kg of water containing 60 g of 6 M HCl. Once these AA were dissolved, the remaining AA were added to the mixture, and after addition of glucose and L-Met (see later), water was 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), and 400 g of acetate/d into the rumen as an energy source.
Treatments were abomasal infusion of 2 amounts (0 or 3 g/d) of L-Met and supplementation with 3 amounts of energy (0, 1.3, and 2.6 Mcal of GE/d; Table 2) in a 2 x 3 factorial design. Methionine was the AA selected for study because it is the first-limiting AA for cattle when metabolizable protein is supplied primarily by ruminal bacteria (Richardson and Hatfield, 1978; Greenwood and Titgemeyer, 2000). The amounts of Met were selected to be in the range of the linear response (0 to 6 g/d) for our experimental model (Campbell et al., 1997; Lambert et al., 2002).
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) allowed increases in energy supply to the animal without increasing ruminal microbial protein synthesis. The VFA were mixed with water such that the total weight of the ruminal infusate was 4 kg/d. A peristaltic pump and polyvinylchloride tubing (2.4 mm i.d.) 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 has been demonstrated in our experimental model (Schroeder et al., 2006) and by others (Hovell et al., 1983; Moloney et al., 1998) that when ruminal adaptation is not required ruminants adapt within 2 d to changes in nutrient supply. Feed samples and refusals (if any) were collected from d 2 through 5 of each period, composited by period, and stored (–20°C) for later analysis. Total urinary (into buckets containing 1.3 L of 1.38 M HCl to keep pH below 3) and fecal outputs were collected daily; samples of urine and feces (1 and 10%, respectively) were saved, composited by period within animal, and stored at –20°C. Before analysis, samples were thawed at room temperature and homogenized. Feed, feed refusals, and fecal samples were partly dried at 55°C for 36 h, air-equilibrated for 36 h, and ground with a Wiley mill to pass a 1-mm screen.
Partly dried diet and fecal samples were analyzed for DM (105°C for 24 h), ash (450°C for 8 h), and NDF with an Ankom-Fiber Analyzer 200 (Ankom-Technology, Fairport, NY) using Na2SO3 and heat-stable amylase. Total N was determined on diet, wet fecal samples, and urinary samples with a Leco FP 2000 N Analyzer (Leco, St. Joseph, MI). Urine samples were analyzed colorimetrically for NH3 (Broderick and Kang, 1980) and urea concentrations (Marsh et al., 1965).
On the last day of each period, whole-body protein turnover was measured by the continuous infusion of labeled Phe (L-2H5-Phe, Cambridge Isotope Laboratories, Andover, MA). Labeled Phe replaced an equal amount (on a molar basis) of the Phe contained in the basal AA mixture, and the labeled mixture was continuously infused (83.3 mg/h) into the abomasum for 10 h. Blood samples were collected 2 h before (background correction) and 10 h after infusion (Lobley et al., 2000; Löest et al., 2002), and enrichment of L-2H5-Phe in plasma was determined through gas chromatography-mass spectrometry (Calder and Smith, 1988) by monitoring ions of weight 336 and 341. The irreversible loss rate for Phe (mmol/h) was estimated as
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This result was converted to whole-body protein flux (WBPF) by dividing by 0.035 (body protein concentration of Phe; Lobley et al., 2000). Whole-body protein synthesis (WBPS) and degradation (WBPD) were calculated (Wessels et al., 1997) from the relationships:
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Thus, WBPS was calculated as
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and WBPD was calculated as
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On d 6 of each period, 10 h after the morning feeding, jugular blood was collected into vacuum tubes (Becton Dickinson, Franklin Lakes, NJ). Blood collected in tubes containing sodium heparin was placed immediately 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, urea, and AA as described by McCuistion et al. (2004). Subsamples of plasma (2 mL) were deproteinized (0.5 mL of 6 M HClO4) and centrifuged (13,800 x g), and the supernatant was neutralized (0.26 mL of 6 M KOH) for ß-hydroxybutyrate analysis (Kientsch-Engel and Siess, 1985).
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 IGF-I and insulin. Concentrations of IGF-I were determined by using an active IGF-I coated-tube immunoradiometric assay kit (intra-assay CV of 8.7% and sensitivity of 5.0 ng/mL; DSL-5600, Diagnostic Systems Laboratories, Webster, TX), and concentrations of insulin were determined with an RIA kit (intra-assay CV of 9.9% and sensitivity of 0.006 ng/mL; DSL-1600, Diagnostic Systems Laboratories).
To characterize the ruminal environment, ruminal fluid was collected after the last period. Ruminal samples were collected from the dorsal, ventral, and caudal areas of the rumen just before, and 4 and 8 h after feeding, and squeezed through 4 layers of cheesecloth. The pH of the ruminal fluid was measured immediately (Orion portable pH meter 230A, Orion Research Inc., Boston, MA), and an 8-mL aliquot was preserved with 2 mL of 25% (wt/vol) metaphosphoric acid and subsequently frozen at –20°C. Samples were centrifuged at 15,000 x g at 4°C for 30 min and analyzed for NH3, as described for urine samples, and for VFA by gas chromatography, as described by Vanzant and Cochran (1994).
Statistical Analyses
Statistical analyses were performed by using the MIXED procedure of SAS System for Windows 8.1 (SAS Inst. Inc., Cary, NC). To allow determination of possible carryover effects, the experimental design was a balanced Latin square. The first analysis included the effects of Met, energy, Met x energy, period, and carryover effect (Morris, 1999); steer was included as a random variable. Seven levels of carryover effect were included in the model (6 treatments plus a pretreatment effect for period 1). The carryover effect was not significant for any variable studied, and it was subsequently excluded from the model (Morris, 1999). Linear and quadratic effects of energy supplementation rate, and their interactions with Met, were tested by single degree of freedom orthogonal contrasts. Treatment means were determined by using the LSMEANS option.
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RESULTS AND DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONIMPLICATIONSLITERATURE CITED |
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). Total VFA concentrations and pH in the rumen were 78 ± 23 mM and 6.4 ± 0.4, respectively.
Nitrogen balance and diet apparent total tract digestibilities are shown in Table 3; the interaction between Met and energy was not significant for any of these variables. Apparent digestibilities of OM and NDF were linearly reduced by energy supplementation, which could be due to the VFA infusion into the rumen affecting ruminal conditions for fiber digestion. These negative effects of energy supplementation on OM digestion slightly reduced basal energy supply by 0.18 and 0.43 Mcal of DE/d. Thus, the planned differences in the amount of energy supplied among treatments were reduced from 1.3 and 2.6 Mcal of GE/d to 1.1 and 2.2 Mcal of GE/d, respectively. It is also possible that the supply of Met from the diet would be decreased by the energy supplements, but reduction in Met supply would strengthen our conclusions regarding responses to energy and Met supplies.
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) or 10 g/d (Lambert et al., 2002). If it is assumed that the empty BW of Holstein steers contains 3.15% N (Fortin et al., 1980), the extra 4.2 g of N/d retained would represent an increase of 133 g/d in ADG. Assuming that retained N is directly converted in protein deposition (N retention x 6.25) and protein for the whole empty body contains 2.0 g of Met/100 g of protein (Ainslie et al., 1993), the calculated efficiency of supplemental Met use was 18% (0.53 g of Met deposited/3 g of Met infused). This efficiency was not significantly affected by energy supplementation.
Energy supplementation linearly increased N retention at both amounts of Met infusion (Table 3). This improvement in N retention was related to a decrease (P < 0.01) in urinary N excretion without changes in fecal N output, and it would represent about 68 and 149 g of added gain/d for the 1.3 and 2.6 Mcal of GE/d treatments, respectively. The increase in N retention in response to energy supplementation at similar (or perhaps lower) levels of Met supply, which were less than the steers’ requirements, demonstrates that there was an increase in the efficiency of Met use as energy supply increased. If the basal absorbable Met supply was 1.0 g/kg of DM intake, as was measured for our diet by Campbell et al. (1997), the basal dietary Met supply was 2.8 g/d. The estimated efficiencies of use of dietary Met when steers did not receive Met supplementation were 96, 106, and 112% for the 0, 1.3, and 2.6 Mcal of GE/d treatments, respectively. Although the absolute values may be overestimated by overestimations of N retention (Gerrits et al., 1996), the relative changes suggest that the overall efficiency of Met use increased with the increase in energy supply. These results indicate that the assumption of a single efficiency of AA use is unlikely to be appropriate for growing cattle across a broad range of energy intakes. Comparing energy supplementation rates similar to those used in our study, Gerrits et al. (1996) also observed increased protein deposition in preruminant calves (80 to 240 kg of BW) when energy supply was increased, even at low protein intakes, supporting the idea that energy supply may affect the efficiency of AA use. Ørskov et al. (1999) observed that steers wholly nourished by intragastric infusion had linearly increased N retention in response to casein infusion, but there was little effect of energy supply when it was more than that needed to reduce ß-hydroxybutyrate concentrations to basal levels. Moreover, infusion of glucose or its precursors seemed to be more important in reducing N excretion than total energy supply, suggesting that dietary energy supply affected protein use when a deficit of glucose existed (Ørskov et al., 1999). In our study, although energy supplementation slightly reduced ß-hydroxybutyrate concentrations in plasma (Table 4), it seems unlikely that our steers were grossly deficient in glucose; plasma glucose concentrations averaged 4.8 mM and were not different among treatments (Table 4). In the current study, we infused a mix of different energy sources (Table 2) with the goal of determining if broad effects of energy supplementation were present. Further research will be needed to evaluate effects of specific energy sources.
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). In our study, as energy supplementation increased, it might be expected that the requirement for Met increased and that, in turn, the supply of Met as a percentage of its requirement decreased. Under these conditions, the improvement in the efficiency of Met use in response to energy supplementation might be a result of the position of our steers on a sigmoidal response of Met use to Met supply, with energy supplementation shifting the steers to a portion of the curve with a steeper slope. Data of Campbell et al. (1997), however, demonstrated a constant efficiency of Met use across amounts of Met supplementation that corresponded to the amounts used in our study. It is possible that the efficiency of Met use is not constant across all levels of Met supply but rather is greater when supplies of Met are less than the amounts absorbed by our control steers.
In our study, WBPS and WBPD increased numerically with Met and energy supplementation (Table 5). The increase for WBPS was numerically greater than for WBPD (Table 5), however, resulting in an increase (P < 0.05) in N retention in response to Met and energy supplementation (Table 3). These results are in agreement with previous studies in which increased total intake (Lobley et al., 2000) or supply of the most limiting AA (Salter et al., 1990; Wessels et al., 1997) resulted in increases in both WBPS and WBPD. Rates of WBPF in our study were greater than those observed in previous studies with growing steers (Wessels et al., 1997; Lobley et al., 2000). This difference may be explained by the fact that we infused the labeled Phe abomasally rather than intravenously and, thus, our values include first-pass metabolism by splanchnic tissues, which accounts for a substantial amount of WBPF (32 to 45%; Reynolds, 2002; Lobley, 2003). Furthermore, our estimates of WBPS (Table 5) were close to the values (2.5 to 2.7 kg/d) reported by Lobley (2003) for steers at the height of productive performance. The amount of protein retained as percentage of the total protein synthesized (Table 5) was not significantly affected by the treatments and was near the value (6%) observed for steers by Lobley et al. (2000).
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). Positive relationships between plasma insulin concentrations and WBPS and WBPD have been observed, but insulin increases WBPS more than WBPD, resulting in a net increase in protein retention (Rooyackers and Nair, 1997). In agreement with those results, energy supplementation increased serum insulin concentrations (quadratic, P < 0.05; Table 4), as well as WBPS, WBPD, and protein retention (Table 5). In response to different sources of energy, Schroeder et al. (2006) observed that protein retention was increased similarly by the sources of energy tested, but that serum insulin concentrations were quite different among sources. This suggests that insulin may be not the most critical regulation of protein deposition in our model. The growth promoting effects of IGF-I, and its positive relationship with protein intake and N retention, have also been demonstrated in growing lambs (Davenport et al., 1995). In our study, the serum concentrations of IGF-I were increased linearly by energy supplementation, but it was not affected by Met supplementation (Table 4). In contrast with our results, it has been observed in preruminant calves (Gerrits et al., 1998) and growing steers (Schroeder et al., 2006) that IGF-I increased with increasing protein supply but not with increasing energy intake, even when energy supplementation resulted in greater protein retention. Therefore, it is difficult to determine if the changes in hormone concentrations mediated the nutritional effects, or reflect changes in the metabolic status due to the alterations in energy and protein supply.
Plasma AA concentrations are presented in Table 4. Supplementation of Met was associated with a significant increase in plasma Met, although the small magnitude of increase indicates that Met supply did not exceed Met requirements. Plasma concentrations of Val, Leu, Ile, Phe, Lys, Trp, Ser, Pro, Asn, and ornithine were decreased by Met supplementation. These changes in plasma AA concentrations may indicate that supplementation with the AA that limited protein synthesis (Met) increased uptake and use of other AA, thereby decreasing their concentrations in the plasma. Previous studies also observed reductions in plasma AA concentrations when supplementation with the most limiting AA was provided to growing steers (Campbell et al., 1997; Wessels et al., 1997). Energy supplementation linearly (Leu) or quadratically (Lys, Asp, Asn, and ornithine) decreased plasma concentrations of some AA, indicating that energy supplementation also increased AA uptake for protein synthesis. Both Glu and Gln plasma concentrations were linearly increased by energy supplementation. In as much as these 2 nonessential AA represent an important energy source for the gastrointestinal tract (Reynolds, 2002; Lobley, 2003), their increase in plasma concentrations may indicate that there was a reduction in the oxidation of these AA as a result of the increased availability of alternative energy sources. This hypothesis is supported by the linear decrease in plasma urea concentrations by the increase in amount of energy supplemented (Table 4). It is also possible that a decrease in the N that passed through the urea cycle could influence concentrations of Glu and Gln, which are important carriers for interorgan transport of N. Plasma Ser concentrations were increased by energy supplementation, likely due to reductions in transsulfuration of Met as energy supplementation increased Met use for protein deposition.
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IMPLICATIONS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONIMPLICATIONSLITERATURE CITED |
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Footnotes |
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2 Corresponding author: etitgeme{at}oznet.ksu.edu
Received for publication September 26, 2005. Accepted for publication January 9, 2006.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONIMPLICATIONSLITERATURE CITED |
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