Effect of Low-Protein Diets on Growth Performance and Body Composition of Broiler Chicks 1,2

Transcription

1 Effect of Low-Protein Diets on Growth Performance and Body Composition of Broiler Chicks 1,2 K. Bregendahl, 3,4 J. L. Sell, and D. R. Zimmerman Department of Animal Science, Iowa State University, Ames, Iowa ABSTRACT Three experiments were conducted to investigate 2 and 3, dietary concentrations of crystalline essential and effects of dietary manipulations to improve nonessential amino acids, respectively, were increased growth performance and whole-body composition of incrementally in the low-protein diets (19 to 20% CP). In broiler chicks fed low-protein diets supplemented with all experiments, chicks fed low-protein diets grew slower, crystalline amino acids. In all experiments, male chicks used feed less efficiently, and retained less N and more (1 d old) were fed a common corn-soybean meal diet ether extract than chicks fed the control diets (P 0.05), (23% CP) for 7 d and subsequently allotted to treatment despite additions of crystalline Gln or Asn and despite diets in a completely randomized design (10 chicks per increased dietary concentrations of crystalline essential floor pen, six replications). Chicks had free access to the isoenergetic diets (3,200 kcal ME n /kg) for 2 wk, after and nonessential amino acids. Chicks fed low-protein which chicks were weighed and then fasted for 24 h, and diets excreted less N (P < 0.001) than did chicks fed the the whole-body DM, N, and ether extract contents of two high-protein diets, and N excretion increased linearly (P chicks per pen (and six baseline chicks) were determined. < 0.001) with N intake. In summary, low-protein diets In Experiment 1, Gln or Asn replaced 1% triammonium citrate in the low-protein diet (19% CP). In Experiments failed to support equal growth performance to that of high-protein control diets. (Key words: broiler chick, low-protein diet, crystalline amino acid, growth performance, body composition) 2002 Poultry Science 81: INTRODUCTION The excretion of N originating from dietary protein is largely responsible for the environmental issues arisen from intensive livestock production (Morse, 1995). In response, dietary means to decrease the impact on the environment of intense livestock production have successfully been implemented; one of which is the partial replacement of intact protein (e.g., soybean meal) with crystalline, free amino acids (AA). Through this replacement, excesses of dietary AA are minimized in relation to their requirement, bringing the dietary protein closer to ideal protein and, in turn, decreasing the dietary CP content. Kerr and Easter (1995) calculated that for each percentage 2002 Poultry Science Association, Inc. Received for publication October 9, Accepted for publication March 7, Journal Paper Number J of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA, Project Number 3812, and supported by Hatch Act and State of Iowa funds. 2 Presented, in part, at the 2001 Midwest and Annual Meetings of the American Society of Animal Science. 3 To whom correspondence should be addressed: kbregend@ uoguelph.ca. 4 Current address: Department of Animal and Poultry Science, Room 255 Animal Science and Nutrition Building, University of Guelph, Guelph, Ontario N1G 2W1, Canada. point decrease in the dietary CP content (with the use of crystalline AA), the amount of excreted N was reduced by 8% in pigs. However, several experiments with broiler chicks (e.g., Fancher and Jensen, 1989a,b,c; Pinchasov et al., 1990; Ferguson et al., 1998; Aletor et al., 2000) and growing pigs (e.g., Kerr et al., 1995; Tuitoek et al., 1997; Knowles et al., 1998) have shown that growth performance and carcass composition become inferior to those of broiler chicks or pigs fed standard high-cp diets when the dietary CP content is lowered by more than three to four percentage points. Although not all experiments support these findings (e.g., Han et al., 1992; Canh et al., 1998), the adverse effects occur even though the low-cp diets meet established AA requirements and have optimal ratios of essential to nonessential AA. Therefore, it is generally not recommended to lower the dietary CP content by more than about three percentage points (Kornegay and Verstegen, 2001; Lewis, 2001). Several studies have investigated potential reasons for the decreased performance. They include modifying the dietary net energy concentrations (Knowles et al., 1998; Leeson et al., 2000), additions of several crystalline nonessential AA (Edmonds et al., 1985; Abbreviation Key: AA = amino acid; EAA = essential amino acid; EE = ether extract; NEAA = nonessential amino acid; SBM = soybean meal. 1156

2 LOW-PROTEIN DIETS 1157 Deschepper and De Groote, 1995; Aletor et al., 2000), decreasing the dietary concentrations of essential AA (EAA) by as much as 13% in relation to recommended concentrations (Pinchasov et al., 1990), increasing the dietary concentrations of EAA by up to 15% in relation to recommended concentrations (Fancher and Jensen, 1989a,c), and manipulating the dietary acid-base balance (Fancher and Jensen, 1989a,c; Waldroup, 2000). However, the reason (or reasons) for the inferior performance has yet to be determined. The objective of the experiments reported herein was to explore possible dietary means by which low-cp diets could be modified to support growth performance and whole-body composition equal to that of chicks fed standard high-protein diets. The effects of dietary additions of Gln and Asn to low-cp diets (Experiment 1) increased dietary concentrations of free EAA (Experiment 2), and increased dietary concentrations of free nonessential AA (NEAA) (Experiment 3) were investigated. MATERIALS AND METHODS All procedures relating to the use of live animals were approved by the Laboratory Animal Resources Committee of Iowa State University. Birds and Whole-Body Analyses We purchased 246, 306, and d-old male broiler chicks (Petersen Hubbard or Hubbard Hubbard) for Experiments 1, 2, and 3, respectively, from a commercial hatchery. 5 The chicks were housed in floor pens ( m) containing pine shavings throughout the experiments and were fed a common diet (similar to the positive control diet in the respective experiments) for 7 d. On Day 7, chicks were allotted to floor pens (10 chicks per pen) on the basis of BW, and each of six replicates of pens was randomly allotted a dietary treatment. Lights were on continuously for the first 10 d posthatching, after which a 17L:7D lighting schedule was maintained for the duration of the experiment. Feed disappearance and BW were measured on Days 14 and 21 posthatching. After the chicks were weighed on Day 21, they were fasted for 24 h (with free access to water) and reweighed on Day 22. Subsequently, two chicks per pen (with a BW close to the pen mean) were selected, euthanized by cervical dislocation, and stored in airtight plastic bags at 20 C for later determination of the whole-body composition. Six chicks, with a BW close to the overall mean, were selected at allotment on Day 7, fasted for 24 h (with free access to water), euthanized by cervical dislocation, and stored in airtight plastic bags at 20 C for later determination of the baseline whole-body composition. 5 Welp Inc., Bancroft, IA. 6 Waring Products Division, New Hartford, CT. 7 U.S. Tecator Inc., Herndon, VA. 8 Laboratory Construction Co., Kansas City, MO. 9 Experiment Station Chemical Laboratories, Columbia, MO. The whole bodies of the euthanized chicks were thawed overnight at room temperature, homogenized, and sampled according to procedures described by Barker and Sell (1994) modified from Sibbald and Wolynetz (1984). The whole body of individual chicks was combined with distilled water (1 BW) in glass beakers, autoclaved for 8 h, and allowed to cool overnight in the autoclave. Weight loss during this process was assumed to be water, which was replaced and the birds subsequently blended in a Waring blender 6 for 2 min after addition of distilled water (2 BW). Duplicate aliquots of 90 to 100 g were dried in plastic weigh boats at 50 C for 3 d, ground with a mortar and pestle, and stored in airtight plastic bags at room temperature for later Kjeldahl N and ether extract (EE) analyses. Whole-body DM was calculated from the dry weight of the ground chicks and records of additions and losses of water (Barker and Sell, 1994). Kjeldahl N and EE were analyzed on pooled samples of the two chicks per pen. The whole-body N content was analyzed in triplicate by the Kjeldahl procedure (Association of Official Analytical Chemists, 1984) using the micro-kjeldahl method on a Kjeltech 1028 distilling unit, 7 and whole-body CP was calculated as Kjeldahl N The whole-body fat content was determined in triplicate as EE according to the Association of Official Analytical Chemists (1984) using a Goldfisch extraction apparatus. 8 Fat and N retention, respectively, were calculated by computing the difference between the whole-body EE or N content at the end of the trial (after 2 wk on test) and the corresponding whole-body baseline content. The apparent N excretion was calculated as the difference between N intake and N retention. The (apparent) N excretion as a percentage of N intake and (apparent) N excretion per gram of N retained were calculated to account for potential differences in N intakes and N retention, respectively, among the dietary treatments. Analyzed N values were used in all calculations. Diets Corn and soybean meal (SBM) were analyzed for CP as Kjeldahl N 6.25 using the micro-kjeldahl method on a Kjeltech 1028 distilling unit 7 and for total AA by ionexchange chromatography 9 after which the contents of true digestible AA were calculated from digestibility coefficients listed by the NRC (1994) and, if unavailable in NRC (1994), the listing by Heartland Lysine (1998) was used. The true digestible AA data were used for formulation of the diets. The broiler requirement for true digestible AA was estimated as 89% (i.e., the average true digestibility coefficient of the AA in corn and SBM) of the recommendation for total AA listed by the NRC (1994). In all experiments, crystalline amino acids were assigned the respective CP and ME values listed by the NRC (1994), and the true digestibility was assumed to be 100% (Izquierdo et al., 1988; Chung and Baker, 1992). All diets were formulated to meet or exceed NRC (1994) recommendations for (true digestible) EAA, minerals, and vitamins; to be isoenergetic (on an ME n basis); and to contain

3 1158 BREGENDAHL ET AL. TABLE 1. Composition of diets in Experiment 1 Ingredient Diet 1A Diet 1B Diet 1C Diet 1D Corn Soybean meal (48%) Soybean oil Dicalcium phosphate Limestone Solka-Floc Potassium carbonate Sodium chloride Sodium carbonate Mineral premix Vitamin premix Triammonium citrate 1.00 L-Asn 1.00 L-Gln 1.00 L-Arg L-Ile L-Val L-Lys HCl DL-Met L-Thr Fiber Sales and Development, Urbana, OH. 2 Supplied per kilogram of diet: manganese, 70 mg; zinc, 90 mg; iron (ferrous sulfate), 60 mg; copper, 12 mg; selenium (sodium selenite), 0.15 mg; sodium chloride, 2.5 g. 3 Supplied per kilogram of diet: vitamin A (retinyl acetate), 8,065 IU; cholecalciferol, 1,580 IU; vitamin E (DLα-tocopheryl acetate), 15 IU; vitamin B 12,16µg; vitamin K (menadione sodium bisulfite), 4 mg; riboflavin, 7.8 mg; pantothenic acid, 12.8 mg; niacin, 75 mg; choline, 509 mg; folic acid, 1.62 mg; biotin, 270 µg. equal concentrations of K, Na, Cl, and crude fiber. The L-Lys HCl, DL-Met, and L-Thr used in the diets were feed grade, whereas all other crystalline AA (in their L- forms), as well as K 2 CO 3 and Na 2 CO 3, were reagent grade (minimum 98% purity) and purchased from Sigma-Aldrich Chemical. 10 The N and CP contents of all diets were determined as described for corn and SBM. Experiment 1. The positive control diet (Diet 1A) was formulated using corn, SBM, and DL-Met (Tables 1 and 2) to meet or exceed the NRC (1994) nutrient recommendations. A negative control diet (Diet 1B) was formulated to contain 18.6% CP using corn, SBM, and free AA to meet the true digestible AA requirement. Diets 1C and 1D were formulated as Diet 1B and contained 1% free L- Gln or L-Asn, respectively, replacing triammonium citrate. A concentration of 1% L-Gln was chosen because Wu et al. (1996) found growth responses to 1% dietary L- Gln in diets for early-weaned pigs. Triammonium citrate, rather than NEAA, was used in Diet 1B as a source of nonspecific N, because AA (including Gln and Asn) have been shown to affect the activity of ornithine decarboxylase in the enterocytes (Minami et al., 1985; Rinehart et al., 1985). Lee and Blair (1972) showed that dietary concentrations of up to 4.4% triammonium citrate could serve as a nonspecific N source for broiler chicks with no adverse effects on growth performance. Experiment 2. The positive control diet (Diet 2A) was formulated using corn, SBM, and DL-Met (Tables 3 and 4) to meet or exceed the NRC (1994) nutrient recommen- 10 St. Louis, MO. dations. A negative control diet (Diet 2B) was formulated to contain 18.5% CP using corn, SBM, and free AA to meet the (calculated) true digestible AA requirement. Diets 2C, 2D, and 2E were formulated to contain, respectively, 15, 30, and 45% more AA from the crystalline sources added in Diet 2B, replacing L-Glu. To ensure that the dietary AA concentrations were not deficient, the requirement for total EAA was set at 105% of the NRC (1994) concentrations, and the requirement for true digestible AA was estimated as 89% of that value. Experiment 3. The positive control diet (Diet 3A) was formulated using corn, SBM, and DL-Met (Tables 5 and 6) to meet or exceed the NRC (1994) nutrient recommendations. A negative control diet (Diet 3B) was formulated to contain 17.6% CP using corn, SBM, and free AA to meet the true digestible AA requirement. Diets 3C, 3D, and 3E were formulated to contain, respectively, 1, 2, and 3% free NEAA from a 1:1 mix of L-Glu and L-Asp, replacing cornstarch. A sixth diet (Diet 3F) was included in the experiment to investigate whether a combination of insufficient amounts of NEAA and less than 100% utilization of free AA was responsible for the lower performance of chicks fed low-cp diets. Hence, Diet 4F contained 2% free NEAA, and the concentrations of free EAA were increased by 45% compared with Diet 4B. To ensure that the dietary AA concentrations were not deficient, the requirement for total AA was set at 105% of the NRC (1994) concentrations and the requirement for true digestible EAA was estimated as 89% of that value. Statistical Analyses Data in all experiments were subjected to ANOVA procedures appropriate for a completely randomized design

4 LOW-PROTEIN DIETS 1159 TABLE 2. Chemical composition of diets in Experiment 1 Item Diet 1A Diet 1B Diet 1C Diet 1D Analyzed values CP (total) Calculated values CP (total) Ether extract Crude fiber Calcium Nonphytate phosphorus Potassium Sodium Chloride True digestible amino acids 1 Arg Gly Gly + Ser His Ile Leu Lys Met Met + Cys Phe Phe + Tyr Ser Thr Trp Val Includes amino acids from intact protein and crystalline sources. Calculated using true digestibility coefficients from NRC (1994) and Heartland Lysine (1998). Crystalline amino acids were assumed 100% true digestible. (Morris, 1999) with six replications. In Experiment 1, treatments means were compared using Fischer s protected least significant difference (StatView, 1999). The contrast of Diet 1A vs. others was used to evaluate the effects of dietary CP concentration (JMP, 2000). In Experiment 2, linear and quadratic contrasts (JMP, 2000) were used to evaluate the effects of increasing amounts of free EAA (i.e., Diets 2B to 2E). In addition, contrasts were used to evaluate the effects of dietary CP concentration (i.e., Diet 2A vs. others). The NEAA added to Diets 3C, 3D, and 3E in Experiment 3 replaced cornstarch not a source of N in Diet 3B. The dietary CP content therefore increased TABLE 3. Composition of diets in Experiment 2 Ingredient Diet 2A Diet 2B Diet 2C Diet 2D Diet 2E Corn Soybean meal (48%) Soybean oil Dicalcium phosphate Limestone Solka-Floc Potassium carbonate Sodium chloride Sodium carbonate Mineral premix Vitamin premix L-Arg L-Glu L-Ile L-Lys HCl DL-Met L-Thr L-Val Fiber Sales and Development, Urbana, OH. 2 Supplied per kilogram of diet: manganese, 70 mg; zinc, 90 mg; iron (ferrous sulfate), 60 mg; copper, 12 mg; selenium (sodium selenite), 0.15 mg; sodium chloride, 2.5 g. 3 Supplied per kilogram of diet: vitamin A (retinyl acetate), 8,065 IU; cholecalciferol, 1,580 IU; vitamin E (DLα-tocopheryl acetate), 15 IU; vitamin B 12,16µg; vitamin K (menadione sodium bisulfite), 4 mg; riboflavin, 7.8 mg; pantothenic acid, 12.8 mg; niacin, 75 mg; choline, 509 mg; folic acid, 1.62 mg; biotin, 270 µg.

5 1160 BREGENDAHL ET AL. TABLE 4. Chemical composition of diets in Experiment 2 Item Diet 2A Diet 2B Diet 2C Diet 2D Diet 2E Analyzed values CP (total) Calculated values CP (total) Ether extract Crude fiber Calcium Nonphytate phosphorus Potassium Sodium Chloride True digestible amino acids 1 Arg Gly Gly + Ser His Ile Leu Lys Met Met + Cys Phe Phe + Tyr Thr Trp Val Amino acids originating from crystalline sources Arg Ile Lys Met Thr Val Includes amino acids from intact protein and crystalline sources. Calculated using true digestibility coefficients from NRC (1994) and Heartland Lysine (1998). Crystalline amino acids were assumed 100% true digestible. TABLE 5. Composition of diets in Experiment 3 Ingredient Diet 3A Diet 3B Diet 3C Diet 3D Diet 3E Diet 3F Corn Cornstarch Soybean meal (48%) Soybean oil Dicalcium phosphate Limestone Solka-Floc Potassium carbonate Sodium chloride Sodium carbonate Mineral premix Vitamin premix L-Arg L-Asp L-Glu L-Ile L-Lys HCl DL-Met L-Thr L-Val Fiber Sales and Development, Urbana, OH. 2 Supplied per kilogram of diet: manganese, 70 mg; zinc, 90 mg; iron (ferrous sulfate), 60 mg; copper, 12 mg; selenium (sodium selenite), 0.15 mg; sodium chloride, 2.5 g. 3 Supplied per kilogram of diet: vitamin A (retinyl acetate), 8,065 IU; cholecalciferol, 1,580 IU; vitamin E (DLα-tocopheryl acetate), 15 IU; vitamin B 12,16µg; vitamin K (menadione sodium bisulfite), 4 mg; riboflavin, 7.8 mg; pantothenic acid, 12.8 mg; niacin, 75 mg; choline, 509 mg; folic acid, 1.62 mg; biotin, 270 µg.

6 LOW-PROTEIN DIETS 1161 TABLE 6. Chemical composition of diets in Experiment 3 Item Diet 3A Diet 3B Diet 3C Diet 3D Diet 3E Diet 3F Analyzed values CP (total) Calculated values CP (total) Ether extract Crude fiber Ca Nonphytate phosphorus Potassium Sodium Chloride True digestible amino acids 1 Arg Gly Gly + Ser His Ile Leu Lys Met Met + Cys Phe Phe + Tyr Thr Trp Val Amino acids originating from crystalline sources Arg Asp Glu Ile Lys Met Thr Val Includes amino acids from intact protein and crystalline sources. Calculated using true digestibility coefficients from NRC (1994) and Heartland Lysine (1998). Crystalline amino acids were assumed 100% true digestible. with higher concentrations of added NEAA. The dietary CP concentrations of Diets 3B, 3C, 3D, and 3E were, nevertheless, lower than the control diet, and so the contrast Diet 3A vs. Diets 3B, 3C, 3D, and 3E was deemed appropriate to evaluate potential differences between the control and low-cp diets. Linear and quadratic contrasts were used to evaluate effects of graded dietary additions of NEAA in Diets 3B, 3C, 3D, and 3E. Furthermore, Diet 3A was compared with Diet 3E (containing the highest amount of added NEAA) using contrasts. Effects of Diet 3F were compared with those of the positive control diet, Diet 3A, using contrasts. In all experiments, pens (each containing 10 chicks) served as experimental units, and P 0.05 was considered significant in all comparisons. Experiment 1 RESULTS Feed consumption data for Diet 1A represented only five pens of chicks. An error occurred in the recorded feed weights of one pen and, therefore, feed consumption data of the pen were deemed unacceptable. However, weight gain of chicks in the affected pen was similar to that of other pens of chicks fed Diet 1A. Thus, weight gain data of chicks in the affected pen are included in the mean representing Diet 1A. Weight gain and feed consumption data are shown in Table 7 and body composition data for the 14-d experiment are shown in Table 8. No differences (P > 0.05) were detected in growth rate, feed consumption, or feed utilization among chicks fed the three low-cp diets, regardless of their content of triammonium citrate, L-Gln, or L-Asn. However, chicks fed any of the three low-cp diets gained less weight (P < 0.05) and utilized the feed less efficiently (P < 0.001) than chicks fed the control diet, Diet 1A. Although there were no differences (P > 0.10) in the whole-body CP concentration, chicks fed Diet 1A had a lower (P 0.01) wholebody concentration of DM and fat than did chicks fed any of the low-cp diets. Moreover, chicks fed Diet 1A retained less fat (P < 0.001) and more N (P < 0.05) than did chicks fed Diets 1B, 1C, and 1D, although the difference in N retention was only significant when the preplanned contrast of Diet 1A vs. others was used. Chicks fed Diet 1A excreted more N (P 0.01) than did chicks fed the low-cp diets, but no differences (P 0.08) were observed among the low-cp diets (Table 9).

7 1162 BREGENDAHL ET AL. TABLE 7. Weight gain and feed consumption of broiler chicks fed diets containing Gln or Asn from 7 to 21 d of age, Experiment 1 Item 1 Diet 1A Diet 1B Diet 1C Diet 1D SEM Weight gain 2 (g/chick) 654 a 631 b 620 b 623 b 7 Feed consumption 2 (g/chick) Gain-to-feed ratio 2 (g/kg) 739 A 685 B 679 B 686 B 4 A,B Least squares means within a row lacking a common superscript differ (P < 0.001). a,b Least squares means within a row lacking a common superscript differ (P < 0.05). 1 Least squares means of six pens, each containing 10 chicks, per dietary treatment. Feed consumption and utilization data for Diet 1A represent only five pens of chicks (see text). Chicks averaged 128 ± 1 g body weight at the start of the experiment. 2 The contrast, Diet 1A vs. others, was significant (P < 0.05). Experiment 2 Weight gain and feed consumption data are shown in Table 10 and body composition data for the 14-d experiment are shown in Table 11. Chicks fed the four low- CP diets (Diets 2B, 2C, 2D, and 2E) gained less weight, consumed more feed, and utilized the feed less efficiently (P 0.001) compared with chicks fed the control diet (Diet 2A). Increasing the dietary concentration of free EAA resulted in a linear (P = 0.01) decrease in feed consumption, but had no linear or quadratic effects (P > 0.10) on weight gain, feed utilization, or whole-body composition. Chicks fed Diet 2A retained more N and less fat (P < 0.001) than did chicks fed any of the low-cp diets. Increased dietary concentrations of free EAA (replacing L- Glu) did not affect (P > 0.10) fat retention, although N retention tended (P = 0.06) to decrease linearly. Chicks fed Diet 2A excreted more N (P < 0.001) than did chicks fed the low-cp diets, but no effects (P > 0.10) on N excretion were observed for the increased dietary concentrations of free EAA (Table 9). Experiment 3 Weight gain and feed consumption data are shown in Table 12 and body composition data for the 14-d experiment are shown in Table 13. Chicks fed the low-cp diets (Diets 3B, 3C, 3D, and 3E) tended (P = 0.07) to gain less weight than chicks fed the positive control diet (Diet 3A). No differences (P > 0.10) were detected in feed consumption, and chicks fed the control diet, therefore, utilized feed more efficiently (P < 0.001) than chicks fed the low- CP diets. There were no linear or quadratic effects (P > 0.10) of increasing the dietary concentrations of free NEAA on weight gain or feed consumption, although feed utilization tended (P = 0.08) to improve linearly. Chicks fed Diets 3B, 3C, 3D, and 3E contained a higher percentage of whole-body fat and a lower percentage of whole-body CP (P < 0.001) than that of chicks fed Diet 3A. Although percent whole-body fat decreased (P < 0.05) linearly with increasing dietary concentrations of free NEAA, the value did not reach that of chicks fed Diet 3A (P < 0.05). Chicks fed Diet 3A retained less fat and more N(P < 0.001) than did chicks fed Diets 3B, 3C, 3D, and 3E. Fat retention (P = 0.05), but not N retention (P > 0.10), was affected by the increase in dietary concentrations of free NEAA, yet fat retention did not (P < 0.05) reach the values obtained with Diet 3A. Chicks fed Diet 3F, a low-cp diet containing a mixture of free EAA (above that added in Diet 3B) and NEAA, tended (P = 0.07) to gain less weight and utilized feed less efficiently (P < 0.001) than did chicks fed Diet 3A. Moreover, chicks fed Diet 3F contained a higher percentage of fat (P < 0.05) and retained less N (P < 0.05) than did chicks fed Diet 3A. No differences in percentages of whole-body CP (P > 0.10) and fat retention (P = 0.08), TABLE 8. Whole-body composition, nitrogen retention, and fat retention of broiler chicks (21 d of age) fed diets containing intact protein or free amino acids, Experiment 1 Item 1 Diet 1A Diet 1B Diet 1C Diet 1D SEM Whole-body composition DM A B B B 0.46 CP Fat A B B B 0.38 (g/chick) Retention 3 Nitrogen Fat A B B B 3.10 A-B Means within a row lacking a common superscript differ (P 0.01). 1 Means of six pens, each containing 10 chicks, per dietary treatment. 2 The contrast, Diet 1A vs. others, was significant (P < 0.05). 3 Chicks contained 3.12 ± 0.01 g nitrogen and 6.83 ± 0.01 g fat on average at the start of the experiment.

8 LOW-PROTEIN DIETS 1163 TABLE 9. Apparent nitrogen excretion of broiler chicks from 7 to 21 d of age Nitrogen excretion Item 1 g g/g of N retained % of N intake Experiment 1 2 Diet 1A A 0.94 A A Diet 1B B 0.74 B B Diet 1C B 0.73 B B Diet 1D B 0.69 B B SEM Experiment 2 3 Diet 2A Diet 2B Diet 2C Diet 2D Diet 2E SEM Experiment 3 3,4,5,6 Diet 3A Diet 3B Diet 3C Diet 3D Diet 3E Diet 3F SEM A,B Means within a row lacking a common superscript differ (P 0.01). 1 Means of six pens, each containing 10 chicks, per dietary treatment. 2 The contrast, Diet 1A vs. Diets 1B, 1C, and 1D, was significant (P < 0.001). 3 The contrast, Diet A vs. Diets B, C, D, and E, was significant (P < 0.001). 4 The linear contrast of Diets 3B, 3C, 3D, and 3E was significant (P < 0.001). 5 The contrast, Diet 3A vs. Diet 3E, was significant (P < 0.001). 6 The contrast, Diet 3A vs. Diet 3F, was significant (P < 0.001). however, were observed between chicks fed the two diets. Although chicks fed the low-cp diets excreted less (P < 0.001) N than did chicks fed the positive control diet (Table 9), a linear (P < 0.001) increase in apparent N excretion with increasing dietary concentrations of NEAA was observed. This increase was evident when measured as total grams of N excreted, as a percentage of N intake, and as grams of N excreted per gram of N retained. Nitrogen excretion by chicks in Experiment 3 (P < 0.001) and chicks in all three experiments (P < 0.001) was directly correlated with N intake (Figures 1 and 2, respectively). DISCUSSION The negative-control, low-cp diets in all three experiments (i.e., the B diets) did not support a growth perfor- FIGURE 1. Linear regression of apparent nitrogen excretion (Y) on nitrogen intake (x) of 7- to 21-d-old broiler chicks, Experiment 3. mance and whole-body composition similar to that of the high-cp control diets (i.e., the A diets) despite meeting or exceeding NRC recommended levels of EAA and containing equal concentrations of Na, Cl, K, and ME n. These findings corroborate results of other studies of low-cp diets (Fancher and Jensen, 1989a,b,c; Pinchasov et al., 1990; Aletor et al., 2000; Leeson et al., 2000). Experiment 1 was designed to investigate whether 1% dietary L-Gln or 1% dietary L-Asn (both replacing 1% triammonium citrate) would improve growth performance and N retention of broiler chicks fed low-cp diets. Decreasing the amount of SBM in low-cp diets also decreases the concentrations of Gln and Asn, normally considered nonessential. However, Gln has been suggested to be a conditionally EAA (Buchman, 1997), because of its role in maintenance of mucosal integrity and growth, and its involvement in the recovery from disease, burns, and starvation (Souba et al., 1990; Cynober, 1991; Souba, 1991). In vitro, Gln stimulates enterocyte protein synthesis (Higashiguchi et al., 1993) and proliferation of enterocytes (Kandil et al., 1995; DeMarco et al., 1999). Moreover, Wu et al. (1996) showed that 1% dietary Gln (but not 0.6%) prevented atrophy of the jejunal villi of pigs weaned early TABLE 10. Weight gain and feed consumption of broiler chicks fed diets containing increasing concentrations of crystalline essential amino acids from 7 to 21 d of age, Experiment 2 Item 1 Diet 2A Diet 2B Diet 2C Diet 2D Diet 2E SEM Weight gain 2 (g/chick) Feed consumed 2,3 (g/chick) Gain-to-feed ratio 2 (g/kg) Means of six pens, each containing 10 chicks, per dietary treatment. Chicks averaged 146 ± 1 g body weight at the start of the experiment. 2 The contrast, Diet 2A vs. others, was significant (P 0.001). 3 The linear contrast of Diets 2B, 2C, 2D, and 2E was significant (P = 0.011).

9 1164 BREGENDAHL ET AL. FIGURE 2. Linear regression of apparent nitrogen excretion (Y) on nitrogen intake (x) of 7- to 21-d-old broiler chicks, pooled data from all experiments (Experiment 1, ; Experiment 2, ; Experiment 3, ). pigs and significantly improved feed utilization in the second week after weaning. In vitro, Gln and Asn but not the structurally similar Glu and Asp (Minami et al., 1985; Rinehart et al., 1985) stimulate ornithine decarboxylase activity in cells of the small intestine (Hayashi, 1989; Kandil et al., 1995; Wang et al., 1996, 1998; Ray et al., 1999), and Gln is involved in maintenance of mitochondrial membrane function and integrity (Ahmad et al., 2001). Thus, chicks fed diets low in Gln or Asn may be at a disadvantage, potentially explaining the poor performance when fed low-cp diets. Yet, adding 1% L-Gln or 1% L-Asn to low-cp diets in Experiment 1 did not improve growth performance, whole-body composition, or fat and N retention significantly. Hence, low-cp diets probably contain sufficient concentrations of Gln and Asn or the two AA can be synthesized endogenously in suffi- cient amounts to sustain normal functions of the enterocytes. In Experiment 2, the effects of increasing the dietary concentrations of EAA above what was needed to meet NRC (1994) requirements for EAA were investigated. Crystalline AA are assumed to be 100% true digestible (Izquierdo et al., 1988; Chung and Baker, 1992) and, therefore, absorbed 100% into the enterocytes. However, experiments have shown differences between the appearance of AA in the portal blood after meals of free AA and intact protein or peptides (Rérat, 1985; Rérat et al., 1992). Potentially, dietary free AA may be preferentially metabolized in the enterocytes, lowering their bioavailability compared to that of AA absorbed in peptide form. Already, there is a substantial metabolism of dietary EAA (Stoll et al., 1998) and NEAA (Windmueller and Spaeth, 1975) in the enterocytes. Moreover, the bioavailability of free AA in low-cp diets may decrease due to Maillard reactions after as little as 1 wk of storage under warm and humid conditions (Mavromichalis and Baker, 2000). In both scenarios (which are not mutually exclusive), increasing the dietary concentrations of dietary free AA may help overcome the potentially low bioavailability of free AA in low-cp diets. Fancher and Jensen (1989a) added crystalline EAA to low-cp diets (approximately 16.7% CP) in amounts exceeding the NRC-recommended EAA levels by up to 15% and observed an improvement in feed utilization by broiler chicks, albeit not to levels observed with the high-protein (18.5% CP) control diet based on intact protein. In Experiment 2 of the current research, dietary concentrations of free EAA were increased in low-cp diets by 15, 30, and 45% above that needed to meet the NRC-recommended concentrations of EAA. In contrast to the findings of Fancher and Jensen (1989a), we did not observe an improvement in feed utilization with the high levels of free EAA, nor was there an improvement in weight gain of the chicks. If, as suggested, AA absorbed in free form are metabolized in the enterocytes to a larger extent than AA absorbed in peptide-form, the capacity for metabolism of free AA in the enterocyte apparently is sufficiently large to metabolize TABLE 11. Whole-body composition, nitrogen retention, and fat retention of broiler chicks (21 d of age) fed diets containing increasing concentrations of crystalline essential amino acids, Experiment 2 Item 1 Diet 2A Diet 2B Diet 2C Diet 2D Diet 2E SEM Whole-body composition DM Fat Crude protein (g/chick) Retention 4 Nitrogen 2, Fat Means of six pens, each containing 10 chicks, per dietary treatment. 2 The contrast, Diet 2A vs. others, was significant (P 0.001). 3 The linear contrast of Diets 2B, 2C, 2D, and 2E tended to be significant (P = 0.06). 4 Chicks contained 3.50 ± 0.01 g nitrogen and ± 0.01 g fat on average at the start of the experiment.

10 LOW-PROTEIN DIETS 1165 TABLE 12. Weight gain and feed consumption of broiler chicks fed diets containing increasing concentrations of crystalline nonessential and essential amino acids from 7 to 21 d of age, Experiment 3 Item 1 Diet 3A Diet 3B Diet 3C Diet 3D Diet 3E Diet 3F SEM Weight gain (g/chick) Feed consumed (g/chick) Gain-to-feed ratio 2,3,4,5 (g/kg) Means of six pens, each containing 10 chicks, per dietary treatment. Chicks averaged 123 ± 1 g body weight at the start of the experiment. 2 The contrast, Diet 3A vs. Diet 3B, 3C, 3D, and 3E, was significant (P < 0.001). 3 The linear contrast of Diets 3B, 3C, 3D, and 3E tended to be significant (P = 0.08). 4 The contrast, Diet 3A vs. Diet 3E, was significant (P < 0.001). 5 The contrast, Diet 3A vs. Diet 3F, was significant (P < 0.001). the relatively high free AA concentration supplied by Diet 2E. Experiment 3 was designed, in part, to investigate whether the lack of response to low-cp diets in Experiments 1 and 2, were due to a deficiency of nonspecific N (i.e., NEAA). Although higher dietary concentrations of CP originating from free NEAA caused a linear decrease in whole-body fat content, no benefits were observed in percent whole-body CP and N retention. Aletor et al. (2000) found that the feed utilization of broilers fed a low- CP diet (15.3% CP) supplemented with NEAA to 22.5% CP was restored to that of the control-fed broilers (22.5% CP), but feed utilization only tended (P = 0.08) to improve in our study with supplemental NEAA. In addition, chicks fed the control diet utilized feed more efficiently than chicks fed the low-cp diet supplemented with 3% free NEAA (i.e., Diets 3A vs. 3E) and there was a linear increase in apparent N excretion with increasing dietary concentrations of NEAA. Evidently, the majority of additional dietary nonessential N was excreted rather than retained by the chicks in Experiment 3 as indicated in Figure 1. The lack of effects on growth performance, whole-body composition, and N retention indicated that the diets in Experiments 2 and 3 contained sufficient nonessential N. In Experiment 2, dietary concentrations of free EAA above what was needed to meet the NRC-recommended concentrations did not significantly improve performance or whole-body composition of chicks fed low-cp diets. Supplementation of low-cp diets with free NEAA did not restore growth performance or whole-body composition to that of control levels (Experiment 3). However, a combination of increased dietary concentrations of free EAA and free NEAA (Diet 3F) seemed to improve performance, although not to levels observed after feeding the control diet, based on intact protein. Growth performance and whole-body composition notwithstanding, chicks fed low-cp diets excreted less N than did chicks fed the high-cp control diets. Because chicks fed the low-cp diets grew slower than chicks fed the high-cp diets, they would excrete more total N than indicated in Table 9 if they were grown to equal BW. However, when the N excretion was related to N retention, the low-cp diets still resulted in a lower N excretion than did the high-cp diets. Thus, N excretion was highly correlated with N intake (Figure 2), which conceptually agrees with the objectives of low-cp diets, namely to supply sufficient EAA to meet the requirements only, in turn decreasing the amounts of excess dietary NEAA. In conclusion, the low-cp diets in all three experiments led to inferior growth performance and N retention, and TABLE 13. Whole-body composition, nitrogen retention, and fat retention of broiler chicks (21 d of age) fed diets containing increasing concentrations of crystalline nonessential and essential amino acids, Experiment 3 Item 1 D Diet 3A Diet 3B Diet 3C Diet 3D Diet 3E Diet 3F SEM Whole-body composition DM 2,3,4, Fat 2,3,4, Crude protein 2, (g/chick) Retention 6 Nitrogen 2,4, Fat 2,3, Means of six pens, each containing 10 chicks, per dietary treatment. 2 The contrast, Diet 3A vs. Diet 3B, 3C, 3D, and 3E, was significant (P < 0.001). 3 The linear contrast of Diets 3B, 3C, 3D, and 3E was significant (P 0.05). 4 The contrast, Diet 3A vs. Diet 3E, was significant (P < 0.05). 5 The contrast, Diet 3A vs. Diet 3F, was significant (P < 0.05). 6 Chicks contained 2.93 ± 0.01 g nitrogen and 6.33 ± 0.02 g fat on average at the start of the experiment.

11 1166 BREGENDAHL ET AL. an increased whole-body fat retention compared with the high-cp control diets. None of the low-cp diets in Experiment 1, 2, or 3 supported growth performance and carcass composition equal to that of chicks fed a standard 23% CP diet. However, chicks fed the low-cp diets excreted less N in the manure, meaning that if minimization of N excretion is valued above that of growth performance and carcass quality, low-cp diets may be fed with success. ACKNOWLEDGMENTS The assistance of the staff of the Poultry Science Center for care of the chicks, the expertise of A. Weisberg in the chemical analyses, and R. Wei for statistical advice are gratefully acknowledged. REFERENCES Ahmad, S., C. W. White, L. Y. Chang, B. K. Schneider, and C. B. Allen Glutamine protects mitochondrial structure and function in oxygen toxicity. Am. J. Physiol. 280:L779 L791. Aletor, V. A., I. I. Hamid, E. Niess, and E. Pfeffer Lowprotein amino acid-supplemented diets in broiler chickens: Effects on performance, carcass characteristics, whole-body composition and efficiencies of nutrient utilisation. J. Sci. Food Agric. 80: Association of Official Analytical Chemists Official Methods of Analysis of the Association of Analytical Chemists. 14th ed. Association of Analytical Chemists, Washington, DC. Barker, D. L., and J. L. Sell Dietary carnitine did not influence performance and carcass composition of broiler chickens and young turkeys fed low- or high-fat diets. Poult. Sci. 73: Buchman, A. L Glutamine: A conditionally required nutrient for the human intestine? Nutrition 13: Canh, T. T., A. J. A. Aarnink, J. B. Schutte, A. Sutton, D. J. Langhout, and M. W. A. Verstegen Dietary protein affects nitrogen excretion and ammonia emission from slurry of growing-finishing pigs. Livest. Prod. Sci. 56: Chung, T. K., and D. H. Baker Apparent and true amino acid digestibility of a crystalline amino acid mixture and of casein comparison of values obtained with ileal-cannulated pigs and cecectomized cockerels. J. Anim. Sci. 70: Cynober, L Ornithine α-ketoglutarate in nutritional support. Nutrition 7: DeMarco, V., K. Dyess, C. M. West, and J. Neu Inhibition of glutamine synthetase decreases proliferation of cultured rat intestinal epithelial cells. J. Nutr. 129: Deschepper, K., and G. De Groote Effect of dietary protein, essential and non-essential amino acids on the performance and carcase composition of male broiler chickens. Br. Poult. Sci. 36: Edmonds, M. S., C. M. Parsons, and D. H. Baker Limiting amino acids in low-protein corn-soybean meal diets fed to growing chicks. Poult. Sci. 64: Fancher, B. I., and L. S. Jensen. 1989a. Dietary protein level and essential amino acid content: Influence upon female broiler performance during the grower period. Poult. Sci. 68: Fancher, B. I., and L. S. Jensen. 1989b. Influence on performance of three to six-week-old broilers of varying dietary protein contents with supplementation of essential amino acid requirements. Poult. Sci. 68: Fancher, B. I., and L. S. Jensen. 1989c. Male broiler performance during the starting and growing periods as affected by dietary protein, essential amino acids, and potassium levels. Poult. Sci. 68: Ferguson, N. S., R. S. Gates, J. L. Taraba, A. H. Cantor, A. J. Pescatore, M. J. Ford, and D. J. Burnham The effect of dietary crude protein on growth, ammonia concentration, and litter composition in broilers. Poult. Sci. 77: Han, Y., H. Suzuki, C. M. Parsons, and D. H. Baker Amino acid fortification of a low-protein corn and soybean meal diet for chicks. Poult. Sci. 71: Hayashi, S.-I Multiple mechanisms for the regulation of mammalian ornithine decarboxylase. Pages in Ornithine Decarboxylase: Biology, Enzymology, and Molecular Genetics. S.-I. Hayashi, ed. Pergamon Pr., Oxford. Heartland Lysine Digestibility of Essential Amino Acids for Poultry and Swine. Version Heartland Lysine, Chicago, IL. Higashiguchi, T., P.-O. Hasselgren, K. Wagner, and J. E. Fischer Effect of glutamine on protein synthesis in isolated intestinal epithelial cells. J. Parenter. Enteral Nutr. 17: Izquierdo, O. A., C. M. Parsons, and D. H. Baker Bioavailability of lysine in L-lysine HCl. J. Anim. Sci. 66: JMP JMP User s Guide. Version SAS Institute Inc., Cary, NC. Kandil, H. M., R. A. Argenzio, W. Chen, H. M. Berschneider, A. D. Stiles, J. K. Westwick, R. A. Rippe, D. A. Brenner, and M. Rhoads L-Glutamine and L-asparagine stimulate ODC activity and proliferation in a porcine jejunal enterocyte line. Am. J. Physiol. 269:G591 G599. Kerr, B. J., and R. A. Easter Effect of feeding reduced protein, amino acid-supplemented diets on nitrogen and energy balance in grower pigs. J. Anim. Sci. 73: Kerr, B. J., F. K. McKeith, and R. A. Easter Effect on performance and carcass characteristics of nursery to finisher pigs fed reduced crude protein, amino acid-supplemented diets. J. Anim. Sci. 73: Knowles, T. A., L. L. Southern, T. D. Bidner, B. J. Kerr, and K. G. Friesen Effect of dietary fiber or fat in low-crude protein, crystalline amino acid-supplemented diets for finishing pigs. J. Anim. Sci. 76: Kornegay, E. T., and M. W. A. Verstegen Swine nutrition and environmental pollution and odor control. Pages in Swine Nutrition. 2nd ed. A. J. Lewis and L. L. Southern, ed. CRC Press LLC, Boca Raton, FL. Lee, D. J. W., and R. Blair Effects on chick growth of adding various non-protein nitrogen sources or dried autoclaved poultry manure to diets containing crystalline essential amino acids. Br. Poult. Sci. 13: Leeson, S., J. D. Summers, and L. J. Caston Net energy to improve pullet growth with low protein amino acid-fortified diets. J. Appl. Poult. Res. 9: Lewis, A. J Amino acids in swine nutrition. Pages in Swine Nutrition. 2nd ed. A. J. Lewis and L. L. Southern, ed. CRC Press LLC, Boca Raton, FL. Mavromichalis, I., and D. H. Baker Effects of pelleting and storage of a complex nursery pig diet on lysine bioavailability. J. Anim. Sci. 78: Minami, H., K.-I. Miyamoto, Y. Fujii, Y. Nakabou, and H. Hagihira Induction of intestinal ornithine decarboxylase by single amino acid feeding. J. Biochem. 98: Morris, T. R Experimental Design and Analysis in Animal Sciences. CAB International, Oxon. Morse, D Environmental considerations of livestock producers. J. Anim. Sci. 73: NRC Nutrient Requirements of Poultry. 9th ed. National Academy Press, Washington, DC. Pinchasov, Y., C. X. Mendonca, and L. S. Jensen Broiler chick response to low protein diets supplemented with synthetic amino acids. Poult. Sci. 69: Ray, R. M., M. J. Viar, T. B. Patel, and L. R. Johnson Interaction of asparagine and EGF in the regulation of orni-

12 LOW-PROTEIN DIETS 1167 thine decarboxylase in IEC-6 cells. Am. J. Physiol. 276:G773 G780. Rérat, A. A Intestinal absorption of end products from digestion of carbohydrates and proteins in the pig. Arch. Tierernähr. 35: Rérat, A., C. Simoes-Nuñes, F. Mendy, P. Vaissade, and P. Vaugelade Splanchnic fluxes of amino acids after duodenal infusion of carbohydrate solutions containing free amino acids or oligopeptides in the non-anaesthetized pig. Br. J. Nutr. 68: Rinehart, Jr., C. A., D. Viceps-Madore, W.-F. Fong, J. G. Ortiz, and E. S. Canellakis The effect of transport system A and N amino acids and of nerve and epidermal growth factors on the induction of ornithine decarboxylase activity. J. Cell. Physiol. 123: Sibbald, I. R., and M. S. Wolynetz Variation among aliquots of entire chicken homogenates. Poult. Sci. 63: Souba, W. W Glutamine: A key substrate for the splanchnic bed. Annu. Rev. Nutr. 11: Souba, W. W., K. Herskowitz, R. M. Salloum, M. K. Chen, and T. R. Austgen Gut glutamine metabolism. J. Parenter. Enteral Nutr. 14:45S 50S. StatView StatView Reference. Version SAS Institute, Cary, NC. Stoll, B., J. Henry, P. J. Reeds, H. Yu, F. Jahoor, and D. G. Burrin Catabolism dominates the first-pass intestinal metabolism of dietary essential amino acids in milk proteinfed piglets. J. Nutr. 128: Tuitoek, J. K., L. G. Young, C. F. M. de Lange, and B. J. Kerr The effect of reducing excess dietary amino acids on growing-finishing pig performance: An evaluation of the ideal protein concept. J. Anim. Sci. 75: Waldroup, P. W Feeding programs for broilers: The challenge of low protein diets. Pages in Proceedings of the Maryland Nutrition Conference for Feed Manufacturers. University of Maryland College Park, College Park, MD. Wang, J. Y., J. Li, A. R. Patel, S. Summers, L. Li, and B. L. Bass Synergistic induction of ornithine decarboxylase by asparagine and gut peptides in intestinal crypt cells. Am. J. Physiol. 274:C1476 C1484. Wang, J.-Y., M. J. Viar, P. M. Blanner, and L. R. Johnson Expression of the ornithine decarboxylase gene in response to asparagine in intestinal epithelial cells. Am. J. Physiol. 271:G164 G171. Windmueller, H. G., and A. E. Spaeth Intestinal metabolism of glutamine and glutamate from the lumen as compared to glutamine from blood. Arch. Biochem. Biophys. 171: Wu, G., S. A. Meier, and D. A. Knabe Dietary glutamine supplementation prevents jejunal atrophy in weaned pigs. J. Nutr. 126: