Maintenance Sulfur Amino Acid Requirements of Young Chicks and Efficiency of Their Use for Accretion of Whole-Body Sulfur Amino Acids and Protein 1

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1 Maintenance Sulfur Amino Acid Requirements of Young Chicks and Efficiency of Their Use for Accretion of Whole-Body Sulfur Amino Acids and Protein 1 HARDY M. EDWARDS, III, and DAVID H. BAKER 2 Department of Animal Sciences and Division of Nutritional Sciences, University of Illinois, Urbana, Illinois ABSTRACT Peterson Hubbard male chicks were used in two bioassays conducted to determine the maintenance requirement and efficiency of utilization of dietary Met and Cys in young chicks. In each assay, chicks were given free access for 10 d to crystalline amino acid (AA) diets containing graded levels of DL-Met (Assay 1) or graded equal levels of DL-Met and L-Cys (Assay 2). Doses of Met represented 5, 10, 40, 55, 70, and 95% of its ideal level in Assay 1, with all other AA maintained at minimized excess levels that were 15% (of ideal) above the various doses of Met, except for Cys, which was maintained at 100% of its ideal level for all treatments. For example, when Met was fed at 40% of its ideal level, all other AA were fed at 55% of their ideal levels, and Cys was fed at 100%. In Assay 2, Met and Cys were fed at equal levels representing 5, 10, 40, 55, 70, and 95% of (Key words: chick, sulfur amino acids, utilization, efficiency, maintenance) INTRODUCTION To develop accurate models of amino acid (AA) requirements, estimates of maintenance requirements and efficiencies of utilizing indispensible AA for whole-body protein accretion are essential. The investigators who have focused on protein and(or) AA accretion over a wide range of dosing levels have generally concluded that protein accretion and AA accretion are linear functions of protein or AA intake, respectively (Hegsted and Neff, 1970; Batterham et al., 1990; Baker, 1991; Chung and Baker, 1992a; Bikker, 1994; Adeola, 1995; Baker et al., 1996; Edwards et al., 1997, 1999). This linearity implies that utilization of a limiting AA above maintenance is constant over a wide range of intakes. The curve-fitting procedures of Gahl et al. (1995), however, were interpreted to indicate diminishing returns in pigs when N or Lys accretion was evaluated as a function of Lys intake. The studies in which Received for publication March 10, Accepted for publication July 1, Funding for this research was provided by the U.S. Poultry and Egg Association, 1530 Cooledge Road, Tucker, GA To whom correspondence should be addressed: d-baker1@uiuc.edu ideal with all other AA maintained at minimized excess levels that were 15% (of ideal) above the various doses of Met + Cys. After 24 h of feed deprivation, chicks were killed for whole-body protein and AA analysis. In Assay 1, Met accretion was a linear (P < 0.01) function of Met intake (r 2 = 0.97). The slope of the Met accretion regression line indicated that 68% of the crystalline Met ingested (above maintenance) was retained. In Assay 2, increases in whole-body protein and whole-body TSAA were linear (P < 0.01) between dosage levels of 5 and 70% of the ideal TSAA level. Slope of the TSAA accretion line between these dose levels indicated that 52% of the TSAA was retained. The TSAA requirement for zero protein accretion was calculated to be 3.2 mg/d or 9.4 mg/d per kg ³ ₄, whereas the TSAA required for zero TSAA accretion was 5.3 mg/d or 15.3 mg/d per kg ³ ₄ Poultry Science 78: efficiencies of AA utilization have been compared suggest that efficiencies are different for each AA, and species differences may exist as well (Heger and Frydrych, 1985; Batterham et al., 1990; Baker, 1991; Chung and Baker, 1992a; Batterham, 1994; Fuller, 1994; Adeola, 1995; Gahl et al., 1997). Batterham (1994) and Adeola (1995) observed that Lys was more efficiently utilized than Thr in pigs, and Gahl et al. (1997) reported the same results in rats. In chicks, Baker et al. (1996), Edwards et al. (1997), Edwards et al. (1999) and H. M. Edwards (unpublished data) found that accretion of Val, Thr, and Lys were 73, 82, and 79% of intake, respectively. In the aforementioned studies of Baker et al. (1996), Edwards et al. (1997), and Edwards et al. (1999), maintenance estimates were determined for Val, Thr, and Lys using whole-body protein and AA analysis of chicks. Previous maintenance AA requirement estimates for poultry were obtained using N balance collection assays in adult cockerels (Leveille and Fisher, 1959, 1960; Leveille et al., 1960; Burnham and Gous, 1992) or chicks (Kim et Abbreviation Key: AA = amino acid. 1418

2 SULFUR AMINO ACID MAINTENANCE REQUIREMENT 1419 al., 1997a,b,c) or a growth model using chicks (Owens et al., 1985). The study reported herein used commercial broiler chicks to determine the efficiency of utilization of Met and the efficiency of utilization and the maintenance requirements of TSAA. These values were determined using whole-body N and AA analysis of chicks fed crystalline AA diets in which absorption efficiencies of AA could be assumed to be 100% (Chung and Baker, 1992b). MATERIALS AND METHODS General Procedures Housing, handling, feeding, and killing procedures were in accord with policies of the University of Illinois Committee on Laboratory Animal Care. In each assay, Peterson Hubbard male broiler chicks 3 were fed a standard 23% CP corn-soybean meal diet until 1600 h on Day 9 posthatching, at which time the feed was removed. On Day 10 following 16 h of feed deprivation, chicks were individually weighed, wing-banded, and randomly alloted to pens such that each pen had a similar initial weight and weight distribution. Four replicate groups of four chicks were used, and chicks were given free access to the experimental diets and water for 10 d. Chicks were maintained on a 24-h constant fluorescent light schedule in thermostatically controlled stainlesssteel batteries 4. Feed was removed after 10 d, and birds were deprived of feed for 24 h (to remove gut fill) before being weighed and killed (CO 2 asphyxiation). Chicks were then immediately frozen at 4 C until later processing. Initial body composition data were obtained from 16 randomly selected chicks at Day 10 posthatching. These chicks had been deprived of feed for 16 h, and their average weight was 150 g, the same average weight as recorded for the chicks assigned to the experimental diets. Diets Chemically defined crystalline AA diets were fed in two bioassays. In Assay 1, chicks were fed six graded doses of Met representing 5, 10, 40, 55, 70, and 95% of the Met requirement for maximal growth. Amino acids other than Met were maintained at 15% excesses relative to Met (expressed in terms of ideal levels), except for Cys, which was maintained at 100% of its ideal level for all treatments. For example, when Met was fed at 40% of its ideal level, all other AA were fed at 55% of their ideal levels, and Cys was fed at 100%. This procedure was used to avoid problems with AA imbalance (Davis and Austic, 1994). Six diets were also fed in Assay 2, each containing 3 Pine Manor, Goshen, IN Petersime Incubator Co., Gettysburg, OH Model 4812, Hobart, Troy, OH Model MM-2M, Cuisinart, Inc., Norwich, CT TABLE 1. Required level and ideal ratio of indispensable amino acids for young chicks Amino acid 1 Required (ideal) level 2 Ideal ratio 3 (%) (% of Lysine) Lysine Arginine Histidine Methionine Cystine Phenylalanine Tyrosine Threonine Leucine Isoleucine Valine Tryptophan Glycine Proline Glutamic acid NA 4 1 All amino acids were provided as L-isomers and were obtained from Nutri-Quest, Chesterfield, MO. Lysine was provided as L-lysine HCl, and histidine was provided as L-histidine HCl H 2 O. The nonamino acid portion of diets consisted of (percentage) soybean oil, 10; solka floc, 3; mineral mixture (Oduho et al., 1994), 5.37; NaHCO 3, 1.5; choline chloride, 0.2; vitamin mixture (Oduho et al., 1994), 0.2; and cornstarch, to 100% diet. Ethoxyquin (125 mg/kg) and DL-α-tocopheryl acetate (20 mg/kg) were also contained in the diet. 2 These required amino acid levels apply to chicks 1 to 3 wk of age fed purified diets containing 14.6% protein equivalent and 3,800 kcal ME/kg. 3 Ideal ratios were based on previous research in our laboratory (Baker and Han, 1994). 4 NA = not applicable. a different dose of Met + Cys (5, 10, 40, 55, 70, and 95% of the ideal level required for maximal weight gain), with AA other than TSAA maintained at 15% above the TSAA levels, expressed in terms of ideal levels. For example, when TSAA were fed at 40% of their ideal level, all other AA were fed at 55% of their ideal levels. Sulfur AA and the other AA were incorporated into the diet at the expense of cornstarch. Table 1 shows the required levels of AA and ideal ratios (percentage of Lys) of the essential AA (Baker and Han, 1994). True digestibility of free AA was considered to be 100% (Chung and Baker, 1992b). The basal diet contained 3,800 kcal ME/kg, and it was assumed that, as Met and other AA were increased at the expense of cornstarch, dietary ME remained essentially constant. Analytical Procedures Frozen chicks from each pen were combined and chopped into small pieces, after which the combined pieces were ground three times. A 6-mm die was used for the first two grindings, and a 3-mm die was used for the third grinding. Solid CO 2 was used to prechill the grinder 5 before each pass through the die to minimize water loss in the samples. Following grinding, a subsample ( 350 g) from each replicate was placed in freezer bags and frozen at 4 C overnight, after which they were freeze-dried. Lyophilized samples were then weighed to determine dry matter, after which they were ground with a food processor 6. Total N was determined by the macro- Kjeldahl procedure (AOAC, 1990) on duplicate freeze-

3 1420 EDWARDS AND BAKER TABLE 2. Accretion of body weight, protein, and methionine in commercial chicks fed graded levels of methionine (Assay 1) 1 Accretion Dietary methionine Intake TSAA in whole-body protein Body Level % of ideal 2 Diet 3 Met 4 weight 4 Protein 4 Met 4 Met Cystine TSAA (%) (g/d) (mg/d) (g/d) (mg/d) (% of CP) Pooled SEM Data are means of four pens of four Peterson Hubbard male chicks during a 10-d assay (10 to 20 d posthatching); average initial weight was 150 g. 2 Cystine was maintained at 100% of its ideal level for all treatments. All amino acids other than methionine and cystine were maintained at a 15% excess level relative to methionine [e.g., when methionine was fed at 40% of its ideal level, all other (nonsulfur) amino acids were fed at 55% of their ideal levels]. 3 Quadratic (P < 0.01) response. 4 Linear (P < 0.01) response. dried samples. Whole-body AA composition was determined on duplicate samples following 24-h hydrolysis with 6 N HCl. For TSAA, samples were preoxidized (using performic acid) prior to hydrolysis. Ion-exchange chromatography 7 was used to quantify AA. Statistical Analysis Results were analyzed using pen means as the experimental unit. The General Linear Models procedure of SAS (SAS Institute, 1990) was used to compute ANOVA. Microsoft Excel 97 was used to derive linear-regression equations and SE values for both Y-intercepts and regression coefficients. In Assay 1, a linear regression equation was derived for Met accretion as a function of Met intake. Protein accretion as a function of Met intake was not derived because Met intake allowed a portion of the excess Cys to be used for accretion of whole-body protein. In Assay 2, linear regression equations were derived for TSAA accretion vs TSAA intake and protein accretion vs TSAA intake. Regression analysis indicated that slope values began to decrease after 70% of the ideal Met level was reached in both Assay 1 and Assay 2. Thus, efficiency calculations and extrapolations to the Y-intercept (for estimation of maintenance requirements) were based on linear regression equations of accretion vs intake values in the TSAA dosing range of 5 to 70% of the ideal level of Met (Assay 1) or TSAA (Assay 2). In Assay 2, the TSAA requirement for maintenance (zero TSAA or protein accretion) was calculated by determining TSAA intake at zero accretion. The estimates of the TSAA maintenance requirement (milligrams/day) were converted to units of intake per kilogram body weight ³ ₄ by assuming that body weight was the average 7 Model 119CL Amion Acid Analyzer, Beckman Instruments, Palo Alto, CA weight (initial + final divided by 2) of the chicks fed TSAA at 95% of its ideal level. Assay 1 RESULTS Methionine intake, accretion of body weight, wholebody protein, and whole-body Met increased linearly (P < 0.01) as dietary Met levels increased from 5 to 95% of ideal levels in Assay 1 (Table 2). Diet intake tended to reach a plateau at about 40% of the ideal Met level, but feed efficiency increased linearly (P < 0.01) up to the highest level of Met fed. Levels of TSAA in whole-body protein were affected little by Met intake. Methionine accretion as a function of Met intake for Met intakes between 5 and 70% of its ideal level (Figure 1) was described by the straight-line equation Y = 3.40 ± ± 0.03 X(r 2 = 0.97). Slope of the Met regression line indicated that the Peterson Hubbard chicks retained 68% of the Met intake above maintenance and below 70% of the Met required for maximal weight gain. Assay 2 Sulfur AA intake, accretion of body weight, wholebody protein, and whole-body TSAA in Peterson Hubbard chicks increased linearly (P < 0.01) as dietary TSAA levels increased from 5 to 95% of ideal levels (Table 3). Diet intake increased linearly (P < 0.01) up to 40% of the ideal TSAA level, after which it tended to reach a plateau. Thus, diet intake of chicks fed TSAA levels representing 40, 55, 70, and 95% of the ideal level did not differ (P > 0.01). Feed efficiency increased linearly (P < 0.01) between the lowest and the highest levels of TSAA fed. Neither Met nor Cys in whole-body protein were affected (P > 0.10) by TSAA intake.

4 SULFUR AMINO ACID MAINTENANCE REQUIREMENT 1421 TABLE 3. Accretion of body weight, protein, and TSAA in commercial chicks fed graded levels of sulfur amino acids (Assay 2) 1 Accretion Dietary TSAA level Intake TSAA in whole-body protein Body Methionine Cystine % of ideal 2 Diet 3 TSAA 4 weight 4 Protein 4 TSAA 4 Methionine Cystine TSAA (%) (g/d) (mg/d) (g/d) (mg/d) (% of CP) Pooled SEM Data are means of four pens of four Peterson Hubbard male chicks during a 10-d assay (10 to 20 d posthatching); average initial weight was 150 g. 2 All amino acids other than methionine and cystine were maintained at a 15% excess level relative to TSAA (e.g., when methionine and cystine were fed at 40% of their ideal levels, all other amino acids were fed at 55% of their ideal levels). 3 Quadratic (P < 0.01) response. 4 Linear (P < 0.01) response. Sulfur AA accretion (Figure 2) as a function of TSAA intake for TSAA intakes between 5 and 70% of its ideal level was described well by the straight line equation Y = 2.74 ± ± 0.02 X (r 2 = 0.97). Slope of this bestfit regression line indicated that the chicks retained 52% of the TSAA intake above maintenance. Protein accretion as a function of TSAA intake (Figure 2) was also described well by the straight-line equation Y = ± ± X (r 2 = 0.96). Extrapolating the linear regression equations for TSAA and protein accretion to the Y intercept so that X at Y zero could be computed resulted in an estimate of the daily TSAA requirement for zero TSAA accretion of 5.3 mg/d (15.3 mg/d per kg body weight ³ ₄ ). The TSAA requirement for zero daily protein accretion FIGURE 1. Best-fit straight-line plot of whole-body Met accretion (Y) as a function of Met intake (X) for chicks fed graded increments of DL-Met (Cys maintained at 100% of ideal for all treatments). Each data point represents the mean value of four male Peterson Hubbard chicks during the period from 10 to 20 d posthatching (Assay 1). was calculated to be 3.2 mg/d (9.4 mg/d per kg body weight ³ ₄ ). DISCUSSION In Assay 1 (Table 2; Figure 1), the efficiency of recovering absorbed Met in whole-body protein was 68% and was constant at all levels of Met intake between 5 and 70% of its requirement for maximal growth. This 68% efficiency of utilization agrees with preliminary work in our laboratory showing the identical efficiency in New Hampshire Columbian chicks. Methionine utilization showed evidence of diminishing returns (i.e., declining efficiency) as its level in the diet was increased over 70% of its ideal level. Previous chick work from our laboratory (Baker et al., 1996; Edwards et al., 1997, 1999) had shown constancy of utilization for Val (73%), Thr (82%), and Lys (79%) at levels up to 95% of their ideal level. The increase in protein accretion in this assay was not only due to increased Met intake but also to increasing levels of Met, allowing a portion of the excess Cys to be used for protein accretion. In Assay 2 (Table 3; Figure 2), the regression of TSAA accretion on TSAA intake (in which equal portions of Met and Cys were incremented simultaneously) resulted in an observed slope value of 52%. This same regression in Assay 1 (assuming a utilizable Cys intake value equal to the intake of Met) resulted in a slope value of 59%. Recalling that Cys was maintained at 100% of its ideal level as Met was incremented upward in Assay 1, one can deduce that more than 50% of the Cys intake was utilized. Leveille et al. (1960) suggested that the maintenance requirement for Cys was less than that for Met, but this was not borne out in our results. It is clear from these bioassays and our previous research (Baker et al., 1996; Edwards et al., 1997, 1999) that different essential AA have different efficiencies of utilization. Our data also agree with other published data showing that TSAA oxidation in chicks is greater than that of

5 1422 EDWARDS AND BAKER FIGURE 2. Best-fit straight-line plots of (top) whole-body TSAA accretion (Y) as a function of TSAA intake (X) and (bottom) wholebody accretion (Y) as a function of TSAA intake (X) for chicks fed equal graded increments of DL-Met + L-Cys. Each data point represents the mean value of four male Peterson Hubbard chicks during the period from 10 to 20 d posthatching (Assay 2). any other AA (Okumura and Muramatsu, 1978; Muramatsu and Okumura, 1980; Webel and Baker, 1999). The estimated maintenance requirement for Met + Cys (Assay 2) was calculated to be 15.3 mg/d per kg body weight 0.75 for broiler chicks. This requirement is in sharp contrast to the Lys maintenance requirement (89 mg/d per kg body weight 0.75 ) estimate by Edwards et al. (1999). Leveille et al. (1960) and Leveille and Fisher (1959) suggested, based on N balance studies, that the TSAA maintenance requirement was at least twice that of the Lys maintenance requirement. It is our belief that using wholebody AA accretion provides a much more defendable estimate of the maintenance requirements for TSAA and Lys accretion than the N balance assays of Leveille et al. (1960) and Leveille and Fisher (1959). With our data, we would also have calculated a TSAA maintenance requirement that was double that of Lys had we used N balance. Baker et al. (1996) and Edwards et al. (1997, 1999) have clearly shown that, at zero protein accretion, negative accretion of Val, Thr, and Lys occurred, similarly suggesting positive accretion of nonessential AA and negative accretion of essential AA. REFERENCES Adeola, O., Dietary lysine and threonine utilization by young pigs: Efficiency for carcass growth. Can. J. Anim. Sci. 75: Association of Official Analytical Chemists, Official Methods of Analysis, 15th ed. AOAC, Washington, D.C. Baker, D. H., Partitioning nutrients for growth and other metabolic functions: Efficiency and priority considerations. Poultry Sci. 70: Baker, D. H., S. R. Fernandez, C. M. Parsons, H. M. Edwards, III, J. L. Emmert, and D. M. Webel, Maintenance requirement for valine and efficiency of its use above maintenance for accretion of whole-body valine and protein in young chicks. J. Nutr. 126: Baker, D. H., and Y. Han, Ideal amino acid profile for broiler chicks during the first three weeks posthatching. Poultry Sci. 73: Batterham, E. S., Ileal digestibilities of amino acids in feedstuffs. Pages in: Amino Acids in Farm Animal Nutrition, J.P.F. D Mello, ed. CAB International, Wallingford, Oxon, U.K. Batterham, E. S., L. M. Andersen, D. R. Baigent, and E. White, Utilization of ileal digestible amino acids by growing pigs: Effects of dietary lysine concentration on efficiency of lysine retention. Br. J. Nutr. 64: Bikker, P., Protein and lipid accretion in body components of growing pigs. Ph.D. thesis, Wageningen Agricultural University, Wageningen, The Netherlands. Burnham, D., and R. M. Gous, Isoleucine requirements of the chicken: Requirements for maintenance. Br. Poult. Sci. 33: Chung, T. K., and D. H. Baker, 1992a. Efficiency of dietary methionine utilization by young pigs. J. Nutr. 122: Chung, T. K., and D. H. Baker, 1992b. Apparent and true digestibility of a crystalline amino acid mixture and casein: Comparison of values with ileal-cannulated pigs and cecectomized cockerals. J. Anim. Sci. 70: Davis, A. J., and R. E. Austic, Dietary amino acid balance and metabolism of the limiting amino acid. Pages in: Proceedings of the Cornell Nutrition Conference. Cornell University Press, Ithaca, NY. Edwards, H. M., III, D. H. Baker, S. R. Fernandez, and C. M. Parsons, Maintenance threonine requirement and efficiency of its use for accretion of whole-body threonine and protein in young chicks. Br. J. Nutr. 78: Edwards, H. M., III, S. R. Fernandez, and D. H. Baker, Maintenance lysine requirement of young chicks and efficiency of its use for accretion of whole-body lysine and protein. Poultry Sci. 78: Fuller, M. F., Amino acid requirements for maintenance, body protein and accretion and reproduction in pigs. Pages in: Amino Acids in Farm Animal Nutrition. J.P.F. D Mello, ed. CAB International, Wallingford, Oxon, U.K. Gahl, M. J., M. D. Finke, T. D. Crenshaw, and N. J. Benevenga, Diminishing returns in weight, nitrogen, and lysine gain of pigs fed six levels of lysine from three supplemental sources. J. Anim. Sci. 73:

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