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ANIMAL NUTRITION |
Department of Animal Science, Michigan State University, East Lansing, MI 48824
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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Key Words: amino acid • muscle growth • pig • insulin-like growth factor-I • insulin
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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). The dietary CP reduction with crystalline AA (CAA) inclusion can effectively reduce N excretion (Le Bellego et al., 2001; Zervas and Zijlstra, 2002; Otto et al., 2003b) and ammonia emission (Otto et al., 2003a) and precisely match the animal’s AA requirements. However, the inconsistency in performance responses in relation to CAA inclusion rate, cost of CAA, and the resulting lack of predictable net benefits have discouraged the systematic adoption of CAA in commercial production (D. W. Rozeboom, Michigan State University, East Lansing, personal communication). For instance, a 3 to 4% unit reduction in dietary CP concentrations with CAA inclusion did not impair N retention (Lopez et al., 1994; Kerr et al., 1995; Le Bellego et al., 2002), whereas greater CP reductions and CAA inclusion rates decreased body protein accretion (Le Bellego et al., 2001; Gomez et al., 2002b; Otto et al., 2003b). An understanding of the underlying physiological mechanisms is critical for effective use of CAA at high inclusion rates, thus contributing to sustainability of swine production.
Differential absorption rate and alteration in AA plasma profile (Yen et al., 2004; Guay et al., 2006) are associated with the level of dietary CAA inclusion and may contribute to a reduced utilization of AA by the whole-body, particularly in the muscle tissue. We hypothesized that CAA used as replacements for limiting indispensable protein-bound AA in reduced-CP diets decrease utilization of AA in the LM.
The specific objectives of this experiment were to determine if the partial replacement of protein-bound AA with CAA 1) modifies free AA pool profile in the LM, and 2) reduces the growth and protein accretion in the LM.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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Four diets were formulated to contain corn and soybean meal in varying ratios as the source of protein-bound AA, as described by Guay et al. (2006). Analyzed CP concentrations were 16.1, 12.8, 10.1, and 7.8% for the 15%, 12% + CAA, 9% + CAA, and 6% + CAA diets, respectively. Analyzed CP concentrations are used throughout the manuscript to identify the respective diets. All diets were formulated to meet the AA requirements on a true ileal digestibility basis for barrows with a predicted lean growth rate of 325 g/d (NRC, 1998). For feed analysis, total feed N, AA, and starch were determined as described by Guay et al. (2006).
Animals and Housing
Pigs were managed throughout the study according to procedures approved by the Michigan State University All-University Committee on Animal Use and Care, as previously described by Guay et al. (2006). Briefly, 20 barrows [Yorkshire x Landrace (44.2 ± 1.3, initial BW)] were selected from 6 litters and placed into individual metabolism crates (1.5 x 1.5 m). Feed was given in 2 equal meals (at 0800 and 1600) per day for the next 13 d. Pigs were weighed at the beginning (d 0) and before euthanasia on d 13.
Blood and Tissue Collection
Blood samples were collected by jugular venipuncture into disposable Vacutainer tubes containing EDTA as anticoagulant (Becton Dickenson, Franklin, NJ) before the morning feeding on d 0, 6, and 12, and 2 h after the morning feeding on d 13, just before euthanasia. After centrifugation (1,800 x g for 15 min), plasma was frozen (–20°C) and later analyzed for concentrations of AA, glucose, insulin, IGF-I, and total protein. Pigs were euthanized immediately after collection with i.v. administration of pentobarbital (86.3 mg/kg of BW), as described by Guay et al. (2006). Liver and right LM were quickly removed and weighed, and samples were excised and frozen at –70°C.
Biochemical Analysis
Liver and LM samples were homogenized with a tissue homogenizer (Ultra-Turrax T27, IKA-Labortechnik, Stenfer, Germany) in distilled water in a ratio of 0.5:2.5 (g/mL). Concentrations of free AA in muscle, liver, and plasma were determined by a precolumn derivatization with phenyl isothiocyanate (PITC) as described by Battaglia et al. (1999), with some modifications. Norleucine (40 µL, 1.25 mM) was added to 200 µL of liver or muscle homogenate, or to 200 µL of plasma, followed by the addition of 1 mL of trifluoroacetic acid (98%):methanol (1:10 ratio).
Liver and muscle homogenates, and plasma samples were centrifuged (5,000 x g) for 10 min, and the supernatant was removed. The supernatant (155 µL) was evaporated to dryness with a centrifuge evaporator (Heto-Holten AS, Gydevang, Denmark) for 3 h. Redrying solution (20 µL of sodium acetate, methanol, and triethylamine in 2:2:1 ratio) was added to each sample to neutralize the pH for optimizing the derivatization reaction between the PITC and AA. Samples were dried with a centrifuge evaporator for 2 h. Derivatizing reagent [20 µL of methanol, water, triethylamine, and PITC (Pierce, Rockford, IL) in a 7:1:1:1 ratio] was added to each sample. After a reaction time of 10 min, samples were dried by evaporative centrifugation for 3 h. Methanol (20 µL) was added to each sample to remove possible PITC residues. Samples were again dried for 3 h by evaporative centrifugation and rehydrated with a diluant solution (200 µL of Pico-Tag solution and methanol in a 4:1 ratio). Samples (20 µL) were then directly injected onto the column (Pico-Tag column, 3.9 x 300 mm, Waters Corporation, Milford, MA) using a Waters 2690 separation module (Waters Corporation).
Amino acids were detected following HPLC separation (Waters 486 absorbance detector, Waters Corporation) at 254 nm. A specific gradient elution was performed at 46°C, using Pico-Tag eluent 1 and 2 (Waters Corporation) as the mobile phase, at a flow rate of 1.0 mL/min. Peak area analyses for AA were performed using the Millennium Chromatography Manager software (Waters Corporation). Peak analysis of Arg in muscle samples could not be done with exactness, thus accurate determination of the Arg concentration was not possible and hence is not reported. Liver and muscle N was measured using a combustion N determinator (Fp-2000, Leco, St. Joseph, MI) with EDTA as the calibration standard. Dry matter content of liver and muscle was determined by drying at 85°C in an airflow oven for 12 h.
Plasma concentration of insulin was measured using an insulin radioimmunoassay kit (ICN Biomedicals Inc., Costa Mesa, CA) with inter and intraassay CV of 7.4 and 9.8%, respectively, and sensitivity of 5.5 µU/mL. Plasma concentration of IGF-I was determined with an IGF radioimmunoassay kit (Diagnostic Systems Laboratories Inc., Webster, TX) with inter- and intraassay CV of 3.6 and 4.2%, respectively, and sensitivity of 6.0 ng/mL. Plasma glucose concentration was analyzed using a peroxidase and glucose oxidase enzyme kit test (Sigma-Aldrich Inc., St. Louis, MO). Plasma urea concentration was determined with a blood urea nitrogen color test kit (Sigma-Aldrich Inc.). Finally, plasma concentration of protein was determined by the method of Lowry et al. (1951), using a detergent-compatible protein assay (BioRad Laboratories, Hercules, CA) and BSA as the standard.
Statistical Analysis
Data were analyzed as a randomized complete block design using the MIXED procedure of SAS (SAS Inst. Inc., Cary, NC). The statistical model included the effects of treatment (diet), initial BW (block), and litter. Means values for treatment effects on growth performance (i.e., ADG and G:F), and the composition (i.e., AA and protein) of liver, muscle, and plasma were compared using orthogonal contrasts (linear and quadratic). Coefficients used for the contrasts were based on the analyzed CP concentration of 16.1, 12.8, 10.1, and 7.8% of the diets and estimated with IML procedure of SAS. For prefeeding concentrations of AA, urea, insulin, and IGF-I on d 6 and 12, day was added as a fourth classification, and the value on d 0 was included as a covariate. Data were analyzed using the repeated option of the MIXED procedure with the autoregressive option.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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The dietary treatments did not have any negative impact on the health status or feed intake of pigs. However, the reduction of the dietary CP concentration linearly decreased ADG and G:F (P < 0.01), and final weight (P = 0.092; Table 1). The reduction in the dietary CP concentration by 3.3, 6, and 8.3% decreased ADG by 11, 17, and 35%, and final weight by 8, 13, and 34%, respectively.
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The sampling day (d 6 or 12) did not have any effect on prefeeding plasma concentrations of urea and AA (P = 0.165); thus only the effects of diets are presented. The reduction in the dietary CP concentration increased prefeeding plasma concentrations of Ala, Gln, and Gly (linear, P < 0.01) and decreased concentrations of Asn and Tyr (linear, P < 0.01; Table 2). Plasma concentrations of Asp and Glu increased initially and decreased thereafter as the dietary CP concentration decreased from 16.1 to 7.8% (quadratic, P < 0.05). For indispensable AA, the reduction in the dietary CP concentration decreased plasma concentrations of Arg, His, Ile, Phe, and Trp (linear, P < 0.01). Plasma concentrations of Thr, Val, and total indispensable AA decreased initially and increased thereafter as the dietary CP concentration decreased from 16.1 to 7.8% (quadratic, P < 0.01). Plasma concentration of Lys appeared to remain constant in pigs fed 16.1, 12.8, and 10.1% CP diets but increased markedly as the dietary CP concentration decreased to 7.8% (linear and quadratic, P < 0.01). With decreasing the dietary CP concentration from 16.1 to 7.8%, total AA concentration increased linearly (P < 0.05) and concentrations of Orn and urea decreased linearly (P < 0.01).
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). Postfeeding plasma concentrations of Gln increased initially and decreased thereafter as the dietary CP concentration decreased from 16.1 to 7.8% (quadratic, P = 0.058). Postfeeding plasma concentrations of other dispensable AA were not affected by the dietary treatments. For indispensable AA, the reduction in the dietary CP concentration decreased postfeeding plasma concentrations of Arg, His, and Leu (linear, P < 0.01) and increased postfeeding concentrations of Lys and Met (linear, P < 0.01). Plasma concentrations of Ile, Phe, Thr, Trp, Val, total AA, and total indispensable AA decreased initially and increased thereafter as the dietary CP concentration decreased from 16.1 to 7.8% (quadratic, P < 0.05). With decreasing the dietary CP concentration from 16.1 to 7.8% CP, concentrations of Orn, Cit, and urea decreased linearly (P < 0.05).
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) and concentrations of insulin (postfeeding: linear, P = 0.072; prefeeding: linear, P < 0.05; Figure 2). Pre- and postfeeding concentrations of plasma glucose and total protein were not affected by the reduction of the dietary CP concentration (results not shown).
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Longissimus muscle weight, weight/BW, DM, CP, and CP/BW decreased with the reduction of the dietary CP concentration (linear, P < 0.05; Table 4). Pigs fed 12.8, 10.1, and 7.8% CP diets had 1, 13, and 18% reductions in the CP content of the LM compared with that of pigs fed 16.1% CP diet. Concentrations of Ala, Asp, Gln, Glu, and Gly in the muscle tissue increased (linear, P < 0.05) and those of Asn and Tyr decreased (linear, P < 0.01) as the dietary CP concentration decreased. Concentrations of free Cys, His, Ile, Phe, Thr, Trp, Val, and total indispensable AA decreased initially and increased thereafter as the dietary CP decreased from 16.1 to 7.8% (quadratic, P < 0.05). Concentrations of Lys appeared to remain constant between pigs fed 16.1 and 12.8% CP diets but increased as the dietary CP concentration decreased from 12.8 to 7.8% (quadratic, P < 0.01). Finally, concentrations of Orn decreased and Cit increased linearly with the reduction of the dietary CP concentration (P < 0.05).
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Liver DM, CP, and CP/BW decreased with the reduction of the dietary CP concentration (linear, P < 0.05; Table 5). Concentrations of free Arg and Cys in liver tissue increased initially and decreased thereafter as the dietary CP concentration decreased from 16.1 to 7.8% (quadratic, P < 0.05). The reduction of the dietary CP concentration linearly increased liver concentration of free Gln (P < 0.05), whereas liver concentrations of free His, Pro, Ser, Tyr, and Orn decreased linearly (P < 0.05).
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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; Kerr et al., 1995; Le Bellego et al., 2002). However, when the dietary CP is reduced by more than 4% units below that of recommended concentrations, other studies (Gomez et al., 2002a; Otto et al., 2003b) have reported decreases in N retention and in carcass and whole body protein accretion. In this study, the growth performance, and the weight and CP content of LM decreased linearly with the reduction in the dietary CP concentration despite the inclusion of CAA to meet the true digestible AA requirements.
In LM, free concentrations of the majority of indispensable AA (His, Ile, Phe, Met, Thr, Trp, and Val) following the morning feeding decreased initially as the dietary CP concentration declined, reflecting the amount of dietary total AA intake. However, as the dietary CP continued to decline with increasing CAA inclusion rates, concentrations of indispensable and dispensable AA dramatically increased and the CP content decreased in muscle tissue, indicating a decrease in utilization of these AA by LM. Although higher concentrations of indispensable AA may also have resulted in part from their rapid rate of absorption compared with that of dietary protein-bound AA shortly after feeding (Williams and Dunkin, 1980; Yen et al., 2004), the fact that muscle extracellular free AA pool increased is indicative of a decrease in intracellular AA uptake for incorporation into proteins. Metges et al. (2000) demonstrated that Leu utilization for whole-body protein synthesis during the postfeeding phase increased in young humans fed intact casein in comparison with that from an equivalent intake of free L-AA. In addition, Phe oxidation increased in growing pigs fed diets containing free Lys once a day compared with that of pigs fed diets containing protein-bound Lys but did not when the diet containing free Lys was fed frequently (Batterham and Bayley, 1989). However, in this study, pigs were only fed twice a day.
Although systemic blood AA profile reflects tissue utilization and metabolism, the change in postfeeding plasma AA profile observed in this study following increasing inclusion rates of CAA may also have resulted from the dietary amount and differential absorption rate of CAA relative to protein-bound AA. The rapid rate of absorption of CAA by the intestinal mucosa cells compared with that of protein-bound AA modifies portal absorption and systemic blood AA profile (Gropper and Acosta, 1991; Yen et al., 2004). In fact, maximal portal appearance of protein-bound Lys was observed 2 h after feeding time (Yen et al., 2004) compared with 30 min for free Lys. In the current study, muscle and blood samples were taken 2 h after feeding time; it is thus unclear whether the resulting change in plasma AA profile in this study reflected the long-term AA utilization by peripheral tissues. Lohrke et al. (2001) have shown that feeding diets with an unbalanced AA profile to young pigs induced upregulation of muscle protein breakdown. In this context, protein degradation in muscle would be associated with higher muscle concentrations of some free dispensable AA, notably Ala, Asp, Gln, and Gly, as observed in this study. In addition, despite the reduction in the dietary intake of these AA, their plasma (Ala, Gln, and Gly) and hepatic (Ala and Gln) concentrations also increased, most likely reflecting increased release by muscle tissue and extraction by hepatic tissue. It is noteworthy to mention, however, that without kinetically defined movements of these AA, one should be cautious in interpreting the results as such. Nonetheless, the increase in free AA concentration observed in LM, which parallels a decrease in the CP content, does indicate that AA utilization decreased as replacement of protein-bound AA with CAA increased.
Although the estimated intakes of total indispensable AA exceeded requirements for the 50-kg barrow, the fact that concentration of free Lys in muscle tissue and plasma appeared to increase earlier compared with that of other AA may also indicate that other indispensable AA such as His, Ile, Thr, Trp, or Val were limiting. Figueroa et al. (2003) suggested Val and His to be the fifth and sixth-limiting AA, respectively, when the dietary CP concentration decreased 4% units below the recommended CP concentration. In fact, results from Figueroa et al. (2003) indicated that 0.33 and 0.50% total His and Val, respectively, in reduced-CP diets sustained a normal ADG and G:F, whereas 0.28 and 0.44% total His and Val did not. In our study, total His and Val concentrations were 0.33 and 0.52%, and 0.26 and 0.51% for 10.1 and 7.8% diets, respectively, indicating that His may have been limiting in the 7.8% CP diet. However, Li et al. (2002) reported that the optimum ratio of dietary Lys to His for 10- to 20-kg piglets was 100:30. In our study, the ratio of true digestible Lys to His was 100:38 for both the 10.1 and 7.8% CP diets.
It is possible that Arg may have been limiting. Post-feeding concentration of plasma Arg (2-h postfeeding on d 13) decreased by 78% as the dietary CP decreased from 16.1 to 7.8%. Arginine can be synthesized from Gln, Glu, and Pro in young pigs (Wu and Knabe, 1995; Wu, 1997), with endogenous synthesis providing more than 50% of the total daily Arg requirement when the diet contains the recommended CP concentration (Wu et al., 1997). In our study, Arg intake decreased by 67% and the synthetic precursors Gln + Glu and Pro decreased by 60 and 52%, respectively, as the dietary CP decreased from 16.1 to 7.8%. Moreover, the lower plasma and liver concentrations of Cit and Orn, 2 substrates for Arg synthesis, indicate that Arg synthesis may have been reduced in pigs fed 10.1 and 7.8% CP diets. Thus, it is possible that the dietary requirements of Arg may be higher in pigs fed reduced-CP diets containing CAA. In addition, although total concentration of AA increased as the dietary CP concentration decreased, the reductions in concentrations of Asn, Ser, and Tyr in muscle and plasma may reflect suboptimal supplies of dispensable AA or N needed to maximize muscle protein deposition and utilization of indispensable AA. In this study, the N of indispensable to dispensable AA ratios were 54:46 and 60:40 in the 10.1 and 7.8% CP diets, respectively, which are different from 45:55 as recommended by Lenis et al. (1999). However, when Otto et al. (2003b) included Glu in similar low-CP diets to achieve a 45:55 N ratio of indispensable to dispensable AA, whole body N retention still decreased.
In our study, unlike LM, liver concentrations of the majority of indispensable AA were not affected by the reduced dietary CP concentration. Increasing Gln and Ala concentrations indicate an increase in hepatic extraction, presumably as a result of increased extracellular concentrations and release of these AA by muscle tissue. As observed for the LM, liver growth and CP content decreased as the dietary CP concentration decreased. This reduction is consistent with Wykes et al. (1996), who reported that hepatic protein synthesis decreased when the dietary CP concentration decreased from 20 to 3%. Although the decrease in ADG in pigs fed the 12.8% CP diet was surprising, LM weight and protein content were not reduced compared with those fed the 16.1% CP diet. Although one should recognize that LM growth and CP content may be insufficient to reflect whole muscle growth and protein accretion, others have also shown that a moderate reduction in the dietary CP does not reduce whole body N retention (Otto et al., 2003b). With this notion in mind, the reduction in ADG with a moderate reduction in the dietary CP from 16.1 to 12.8% most likely reflected a reduction in liver and other splanchnic organ weights. Others studies have reported a reduction in the weight of liver, pancreas, and digestive tract by feeding reduced-CP + CAA diets (Le Bellego et al., 2002; Gomez et al., 2002b).
In addition to nutritional factors, anabolic hormone responses to the reduction of the dietary CP concentration may contribute to a decrease in AA utilization. In young pigs, insulin infusion increases protein synthesis in various skeletal muscles (Davis et al., 2002), whereas somatotropin increases concentration of plasma IGF-I and subsequent protein deposition through suppression of protein degradation and AA catabolism (Vann et al., 2000; Bush et al., 2002). In this study, the reduction of the dietary CP concentration decreased linearly concentrations of plasma insulin and IGF-I. These findings are consistent with results of Caperna et al. (1990), who reported that reduction of dietary CP concentration from 27 to 11% decreased concentrations of plasma insulin and IGF-I. Data reported herein do indicate a relationship between reductions of whole-body and LM growth and plasma insulin and IGF-I responses to the reduction in the dietary CP concentration, despite the inclusion of CAA.
In summary, reductions in protein-bound AA from corn and soybean meal-based diets reduced whole-body and LM growth, altered free AA pool profile in LM, and decreased concentrations of plasma insulin and IGF-I. The curvilinear nature of the change in free AA profile in muscle tissue and the parallel changes in weight and CP content in LM indicate that utilization of indispensable AA by LM decreased when the replacement of protein-bound AA with CAA increased to the highest rate.
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IMPLICATIONS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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Footnotes |
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2 This study was supported by the Michigan Agric. Exp. Stn. F. Guay was supported by a postdoctoral fellowship from the ‘Fond pour la Formation des Chercheurs et l’Aide à la recherche du Québec’ (FCAR). The authors wish to thank J. Moore, J. Pérez Laspiur, P. Ku, and C. Wickens for their assistance in tissue collection and Duane E. Ullrey for reviewing the manuscript.
3 Current address: Département des sciences animales, Faculté des sciences de l’agriculture et de l’alimentation, Pavillon Paul-Comtois, Université Laval, Quebec, QC G1K 7P4, Canada.
4 Corresponding author: trottier{at}msu.edu
Received for publication September 28, 2005. Accepted for publication May 30, 2006.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONIMPLICATIONSLITERATURE CITED |
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Linear, P = 0.054; *linear, P < 0.05; n = 5.