The use of free amino acids allows formulating very low crude protein diets for piglets 1

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1 Published November 24, 2014 The use of free amino acids allows formulating very low crude protein diets for piglets 1 M. Gloaguen,* N. Le Floc h,* E. Corrent, Y. Primot, and J. van Milgen* 2 *INRA, UMR1348 PEGASE, F Saint-Gilles, France; Agrocampus Ouest, UMR1348 PEGASE, F Rennes, France; and AJINOMOTO EUROLYSINE S.A.S., F Paris Cedex 17, France Abstract: Reducing the dietary CP level with free AA supplementation allows reduction of N excretion and the risk of gut disorders while maintaining performance of pigs. We have recently reevaluated the Val, Ile, His, and Leu requirements in pigs, which enables formulating very low CP diets. However, requirements for undifferentiated N, and thus the extent to which the dietary CP content can be reduced, are unknown. Two experiments were conducted to test the effect of reducing the dietary CP content to meet an ideal AA profile on N retention and performance in 10- to 20-kg pigs. In the first experiment, we measured N retention in 6 blocks of 4 pigs each, all receiving diets with 1.15% standardized ileal digestible (SID) Lys. Each pig within a block was allotted to different levels of dietary CP (19.7%, 16.8%, 14.0%, and 12.7%). The reduction of CP content from 19.7% to 16.8% had no impact on N retention and decreased N excretion by 29%. A further decrease in CP content to 14.0% and 12.7% resulted in a reduced N retention (P < 0.01). In the second experiment, we measured performance in 14 groups of 6 pigs each, allotted to 6 levels of dietary CP with 1.00% SID Lys with soybean meal (17.6%, 15.6%, 13.5%, and 11.8%) or without soybean meal (14.0% and 13.0%). Decreasing the dietary CP content from 17.6% to 13.5% had no effect on performance. Daily gain was reduced in pigs receiving the 11.8% CP diet (P < 0.01). Feeding the diet formulated without soybean meal with 13.0% CP content resulted in reduced feed efficiency, but the addition of Glu to increase the CP content from 13.0% to 14.0% restored performance (P < 0.01). In conclusion, the use of l-val, l-leu, l-ile, l-his, and l-phe enables a 4% unit reduction in the CP content relative to SID Lys in diets for pigs. Soybean meal can be totally replaced using cereals and free AA. However, a further reduction in dietary CP:Lys level below 13.5% reduces feed efficiency, indicating that the supply of N for the synthesis of dispensable AA may be a limiting factor for the performance of pigs. Key words: amino acid, low crude protein, nitrogen balance, performance, pig 2014 American Society of Animal Science. All rights reserved. J. Anim. Sci : doi: /jas INTRODUCTION 1 The authors wish to thank A. Meresse, Y. Jaguelin, A. Starck, F. Guérin, P. Touanel, G. Guillemois, and J. F. Rouaud at INRA and Agrocampus Quest and M. Eudaimon at AJINOMOTO EUROLYSINE for their skillful technical assistance and helpful collaboration. 2 Corresponding author: jaap.vanmilgen@rennes.inra.fr Received March 25, Accepted December 6, Reducing the dietary CP content and using free AA enable improving the efficiency of N utilization while maintaining performance of pigs as long as the AA requirements are met. Because of a low feed intake capacity and a high potential for protein deposition, piglets are offered diets with a high CP content. However, undigested proteins contribute to the proliferation of pathogenic bacteria in the gut, increasing the risk of digestive disorders (Ball and Aherne, 1987). Reducing the dietary CP content improves the health of piglets by reducing the incidence of diarrhea (Wellock et al., 2008; Heo et al., 2009). Lysine, Thr, Met, and Trp are the most limiting AA for growth in cereal soybean meal based diets, and the supplementation of these AA in free form allows for a reduction of the dietary CP content by approximately 4% units (Figueroa et al., 2003; Kerr et al., 2003). The potential to further reduce the dietary CP level may be limited by knowledge of the requirements for the next-limiting AA. Figueroa et al. (2003) suggested that Val, Ile, and His are the next-limiting AA for growth in pigs. The requirements for these AA have been recently reevaluated in 10- to 20-kg pigs (Barea et al., 2009; Gloaguen et al., 2012; van Milgen et al., 2012), making it possible to further balance the

2 638 Gloaguen et al. dietary AA profile to reduce the dietary N content. The objective of this study is to evaluate the extent to which the dietary CP content can be reduced by substituting soybean meal by wheat, barley, corn, and free AA to meet an ideal AA profile without reducing N retention and performance of pigs. MATERIALS AND METHODS Experimental procedures and animal care were performed according to French legislation at the time of the experiments (2011 and 2012). Authorization to experiment on living animals was provided by the French Ministry of Agriculture (certificate numbers 7704 and 7719 for J. van Milgen and N. Le Floc h, respectively). Animals, Diets, and Experimental Design Two experiments were performed with 6-wk-old barrows and female pigs (Pietrain [Large White Landrace]) originating from the experimental herd of INRA (St-Gilles, France). After weaning at 4 wk of age, pigs received a commercial diet and were accustomed to housing by 2 in cages ( cm) equipped with low-pressure water nipples offering free access to water. At 5 wk of age, pigs were blocked by sex, BW, and origin (siblings or half siblings) and housed individually. Pigs within a block were allotted to different treatments. The ambient temperature was maintained at 28 C the first week after weaning and was decreased by 1 C per week thereafter. Pigs were weighed at the beginning and at the end of the experimental period after an overnight fast for calculation of ADG. Feed intake and feed refusals were measured weekly. Feed samples were collected weekly to determine the DM content for calculation of ADFI. Those samples were pooled at the end of the experimental period for analysis. The objective of diet formulation was to minimize the dietary CP content by substituting soybean meal by wheat, barley, corn, and free AA (Table 1). Levels of AA supplementation were calculated to cover the requirements of standardized ileal digestible (SID) relative to Lys according to Sève (1994) for Met (30%), Met + Cys (60%), and Thr (65%); Simongiovanni et al. (2012) for Trp (22%); Barea et al. (2009) for Val (70%); Gloaguen et al. (2012) for Ile (51%), Leu (100%), and His (32%); and NRC (1998) for Arg (42%). In Exp. 1, the effect of reducing the dietary CP content on N balance at constant feed intake was evaluated. Twenty-four barrows (11.7 ± 1.1 kg) were allotted to 1 of 4 diets with CP contents of 19.6%, 16.8%, 14.0%, and 12.7% (Table 1). Diets provided 1.15% SID Lys, and the NE content ranged from 9.9 to 10.4 MJ/kg. The 19.6% CP diet was used from 10 to 14 d postweaning to replace the prestarter diet. Three days before the beginning of the experiment, the amount of feed offered was 3.5% of BW (approximately 80% of ad libitum) and then adjusted every 3 d according to the anticipated increase in BW (Gloaguen et al., 2011). Nitrogen balance was determined during 2 wk period. Feces and urine were collected daily. Feces were weighed, homogenized, and pooled by week, and subsamples were freeze-dried for analysis. Urine was collected in a 10% solution of H 2 SO 4 (approximately 10 ml/l urine), pooled by week, and weighed, and a sample was taken for analysis. A second experiment was performed to study the effect of reducing the dietary CP content on performance of pigs offered feed ad libitum. Eighty-four piglets (12.7 ± 1.4 kg) were allotted to 1 of 6 diets with CP contents relative to SID Lys (CP:Lys) of 17.6%, 15.6%, 13.5%, 11.8%, 13.0%, and 14.0%. Diets were sublimiting in Lys at 1.00% SID (Table 1) to express the AA and CP supply relative to Lys (Boisen et al., 2000; Barea et al., 2009). From 17.6% to 13.5% CP:Lys, l-lys, l-thr, dl-met, l-trp, l-val, l-ile, l-leu, l-his, and l-phe were used (if necessary) to satisfy the AA requirements. The diet with 13.0% CP:Lys was formulated without soybean meal and contained 40% wheat and 28% barley. For this diet, l-arg was added to meet the Arg requirement, and l-pro and l-gly were added to provide the same level of SID Pro:Lys and (Gly + Ser):Lys as the 15.6% CP:Lys diet. The l-glu was used as a N source to attain a 13.0% CP:Lys level. In the last treatment, the effect of N was tested by the addition of l-glu to attain 14.0% CP:Lys. Diets were formulated to provide 9.2 MJ NE/kg, 4.0 g digestible P/kg, and 2.9 g Ca/g digestible P. Because a low electrolyte balance can affect voluntary feed intake (Patience et al., 1987), sodium bicarbonate was included in the diets to maintain an arbitrary electrolyte balance of 180 meq/kg (Apper-Bossard et al., 2009). The prestarter diet was gradually replaced by the experimental diets at 10 d postweaning, so that from 12 d postweaning onward, pigs were only offered the experimental diets. The experiment lasted 21 d. Chemical Analyses Chemical analyses of diets have been described in detail by Gloaguen et al. (2011). Briefly, the GE content was measured with an adiabatic bomb calorimeter (ISO 9831:1998; C2000 and C5000; IKA, Staufen, Germany). Dietary CP content (N 6.25) and N content in feces and urine were obtained after N analysis according to the Dumas procedure using a rapid N cube (Elementar France, Villeurbanne, France; method NF V18-120; AFNOR, 1997). Amino acids from dietary proteins were released by acid hydrolysis for 23 h at 110 C under reflux. Total Met and Cys were hydrolyzed after oxidation

3 Low crude protein diets for piglets 639 Table 1. Composition of experimental diets (as-fed basis) Item Ingredients, % Exp. 1 Exp. 2 Cereals soybean meal free AA Cereals free AA 19.7% CP % CP % CP % CP % CP % CP % CP % CP % CP % CP 1 Wheat Barley Corn Soybean meal Corn starch Sunflower oil l-lys HCl l-thr l-trp dl-met l-val l-his l-ile l-leu l-phe l-arg l-glu l-gly l-pro Phytase Sodium bicarbonate Salt Calcium carbonate Dicalcium phosphate Vitamin and mineral premix Analyzed composition, 3 % CP Ether extract Crude Fiber NDF ADF ADL Lys Thr Met Met + Cys Trp Ile Val Leu His Phe Tyr Arg Ala Asp Glu Gly Ser Pro Calculated composition 3 NE, 4 MJ/kg SID Lys, 5 % Dig N:SID Lys, 5 % EB 5, meq/kg Expected CP content. 2 Supplied per kilogram of diet (as-fed basis): vitamin A, 10,000 IU; vitamin D 3, 2,000 IU; vitamin E, 20 mg; vitamin K 3, 2 mg; vitamin B 1, 2 mg; vitamin B 2, 5 mg; niacin, 20 mg; pantothenic acid, 10 mg; vitamin B 6, 5 mg; biotin, 0.2 mg; folic acid, 1 mg; vitamin B 12, 0.03 mg; chloride de choline, 600 mg; vitamin, 40 mg; Fe as iron sulfate, 100 mg; Cu as copper sulfate, 20 mg; Zn as zinc oxide, 100 mg; Mn as manganese oxide, 40 mg; I as calcium iodate, 0.6 mg; Se as sodium selenite, 0.3 mg; and Co as cobalt carbonate, 1 mg. 3 Adjusted for 87.3% DM. 4 Values for NE were calculated according to Noblet et al. (1994). 5 SID Lys = standardized ileal digestible Lys; Dig N = fecal digestible N, calculated from the analyzed Lys and N content, the fecal N digestibility, and ileal Lys digestibility of the feed ingredients (Sauvant et al., 2004); EB = electrolyte balance calculated from the feed ingredients (Sauvant et al., 2004).

4 640 Gloaguen et al. with performic acid. Total AA content in the diets was analyzed by ion exchange chromatography and ninhydrin derivatization (JLC-500/V AminoTac Amino Acid Analyzer; Jeol, Croissy-sur-Seine, France; method NF EN ISO 13903:2005). Total Trp was analyzed by HPLC with a fluorescence detector (RF 10AXL; Shimadzu, Bonneuil sur Marne, France) after an alkaline hydrolysis with barium hydroxide. Statistical Analysis Data were analyzed using the PROC MIXED procedure (SAS Inst. Inc., Cary, NC) with diet as fixed effect and block as a random effect. The normality of the data and homogeneity of the variances among treatments were tested and confirmed using the UNIVARIATE and MIXED procedures, respectively. Results are reported as least squares means. Probabilities less than 0.05 were considered as significant. RESULTS All pigs appeared healthy throughout the experiment. The results of nutrient analyses of the diets are presented in Table 1. Because of an inappropriate constraint for the minimum level of Trp in the 17.6% CP:Lys diet in Exp. 2, 0.42% l-trp was included in the diet, which resulted in an analyzed SID Trp:Lys of 55% instead of an expected value of 22%. The analyzed AA and CP values agreed closely with the anticipated values. Therefore, the anticipated values were used for all calculations. In Exp. 1, there were no differences in daily feed intake among treatments. Reducing dietary CP level from 19.6% to 16.8% had no effect on performance and retained N (Table 2). Pigs offered the 16.8% CP diet retained 71.0% of the ingested N and 83.8% of the absorbed N. From 19.6% to 16.8% CP, N excretion was reduced by 29%. Feed efficiency, ADG, and retained N of pigs offered the 14.0% and the 12.7% CP diets were lower than for pigs offered diets with a greater CP content (P < 0.05). In Exp. 2, dietary CP:Lys levels did not affect ADFI (Table 3). Lowering the dietary CP:Lys content from 17.6% to 13.5% of diets formulated with soybean meal had no effect on performance. The ADG and G:F of pigs offered the 11.8% CP:Lys were lower (P < 0.05) than those observed in pigs fed diets with greater CP:Lys contents. Feeding the 13.0% CP:Lys diet formulated without soybean meal resulted in a decreased feed efficiency (P < 0.05). Supplementation of this diet with l-glu to attain 14.0% dietary CP:Lys restored feed efficiency (P < 0.05). Table 2. Effect of reducing the dietary CP content on the N balance in 10- to 20-kg pigs (Exp. 1) 1 CP, % Item RSD 2 P-value Initial BW, kg Final BW, kg ADFI, 3 g/d ADG, 3 g/d 375 a 364 a 323 b 305 b 41 <0.01 G:F, 3 g/g 0.73 a 0.69 a,b 0.62 bc 0.58 c 0.10 <0.01 Nitrogen balance Ingested, g/d 15.5 a 13.8 b 11.5 c 10.5 d 0.8 <0.01 Absorbed, g/d 13.3 a 11.7 b 9.7 c 9.0 d 0.7 <0.01 Retained, g/d 9.9 a 9.8 a 8.3 b 7.5 c 0.8 <0.01 Digestibility, % Retained, % of intake 63.9 a 71.0 b 72.2 b 71.4 b 4.1 <0.01 Retained, % of absorbed 74.4 a 83.8 b 85.6 b 83.3 b 3.7 <0.01 a c Within a row, values with different superscripts are different (P < 0.05). 1 Data are presented as least squares means with 6 pigs per treatment. 2 RSD = residual SD, which is the root-mean-square of the error that applies to the whole model. 3 Adjusted for 87.3% DM. DISCUSSION The experiments in this study were conducted to determine the extent to which the CP content of the diet can be reduced without affecting N retention and performance in pigs when the AA requirements are satisfied. Diets were formulated to match or exceed the ideal AA levels. However, a feed formulation error resulted in 55% SID Trp:Lys in the 17.6% CP:Lys diet in Exp. 2. An AA imbalance may reduce feed intake, especially when associated with an AA deficiency (Harper et al., 1970). In the present study, the performance of pigs fed the diet with excess Trp was similar to those fed the other diets (except for the diet with the lowest level of CP) and in line with results of other studies we conducted on AA requirements (Gloaguen et al., 2011, 2012). Consequently, it appears unlikely that the excess Trp supply resulted in an AA imbalance affecting performance. It is well known that Lys, Thr, Met, and Trp are the first-limiting AA for growth in cereal soybean meal diets and that the use of these AA in free form enables reducing dietary CP level (Figueroa et al., 2003; Kerr et al., 2003). The results of this study show that the CP:Lys content of a cereal soybean meal diet, supplemented with free Lys, Thr, Met, and Trp, can be further decreased by 4% units with the addition of free Val, Ile, Leu, His, and Phe without affecting the performance in 10- to 20-kg pigs. Valine has been identified as the fifth-limiting AA after Lys, Thr, Met, and Trp (Mavromichalis et al., 1998; Figueroa et al., 2003; Lordelo et al., 2008). In the present study, the addition of l-val is required below 17.6% dietary CP:Lys and

5 Low crude protein diets for piglets 641 Table 3. Effect of reducing the dietary CP content on performance in 10- to 20-kg pigs (Exp. 2) 1 Cereals soybean meal free AA Cereals free AA Item 17.6% CP 15.6% CP 13.5% CP 11.8% CP 13.0% CP 14.0% CP RSD 2 P-value Initial BW, kg Final BW, kg 22.2 a 22.2 a 21.9 a 20.1 b 21.8 a 22.3 a 1.7 <0.01 ADFI, 3 g/d ADG, 3 g/d 450 a 454 a 442 a 358 b 420 a 451 a 67 <0.01 G:F, 3 g/g 0.59 a 0.59 a 0.57 a 0.49 b 0.52 b 0.58 a 0.06 <0.01 a,b Within a row, values with different superscripts are different (P < 0.05). 1 Data are presented as least squares means with 14 pigs per treatment. 2 RSD = residual SD, which is the root-mean-square of the error that applies to the whole model. 3 Adjusted for 87.3% DM. allows reducing dietary CP:Lys by 2.0% units in Exp. 2. Others have also shown that pigs fed low-cp diets (approximately 17% CP) supplemented with free AA including Val or Val + Ile were able to maintain performance (Le Bellego and Noblet, 2002; Lordelo et al., 2008). In Exp. 1, a decrease in the dietary CP level to 14.0% and 12.7%, with the addition of l-his, l-ile, l-leu, and l-phe, reduced N retention, indicating the existence of 1 or more limiting factors for protein deposition. In Exp. 2, where pigs were fed ad libitum, the addition of these AA allows reducing dietary CP:Lys from 15.6% to 13.5% without affecting performance. The CP:Lys content of a cereal soybean meal diet can be reduced from 17.6% to 13.5% with the supplementation of free Val, Ile, Leu, His, and Phe. A further reduction in dietary CP:Lys content, below 13.5%, reduces N retention, feed efficiency, and growth. The complete substitution of soybean meal by cereals and free AA was studied in Exp. 2. All indispensable AA (IAA) were required in free form in the diets to cover the ideal AA profile for these AA. The cereals and free IAA provided 11.1% CP:Lys. To provide a sufficient amount of dispensable AA (DAA), the diet was supplemented with l-pro and l-gly, according to the complete purified diet for pigs developed by Chung and Baker (1991). The l-glu was used to achieve a CP:Lys content of 13.0%. However, the ingestion of the 13.0% CP:Lys diet resulted in a reduced feed efficiency compared with pigs fed higher CP:Lys levels. This reduced performance may result from a deficiency of dietary N because the addition of l-glu to attain 14.0% CP:Lys restored feed efficiency. The ADFI was not affected, indicating that a N deficiency does not activate mechanisms involved in feed intake regulation. These results also indicate that in the 14.0% CP:Lys diet, in which 78% of the Lys requirement is supplied by free Lys, the efficiency of free AA utilization is not lower than that of protein-bound AA in pigs offered feed ad libitum. Free AA are absorbed more rapidly than protein-bound AA, which may lead to a greater oxidation of AA (Metges et al., 2000; Yen et al., 2004). This can explain why the growth response of pigs fed once daily decreases when the diet contains free Lys compared with those fed a diet with protein-bound Lys or with a frequent feeding (Batterham and Bayley, 1989). However, in agreement with our results, Le Bellego et al. (2001) showed that the N utilization is not affected in low-cp diets when at least 2 meals are fed. When the dietary CP content was reduced by 2.9% units from 19.6% to 16.8%, N excretion decreased by 29%. This confirms that N excretion is reduced by 10% for each percentage unit reduction in low-cp diets (Dourmad et al., 1993; Canh et al., 1998). Therefore, when the dietary CP level is reduced by 4% units, N excretion may decrease by at least 40%. The N digestibility was not affected by dietary CP reduction, which indicates that excess N from dietary AA was mainly excreted in urine. In Exp. 1, pigs offered the 16.8% CP diet efficiently used dietary N and retained 84% of the absorbed N. A lower value has been reported by Le Bellego et al. (2001) in growing pigs (65 kg), which retained 73% of the absorbed N when fed a 12.3% CP diet supplemented with l-lys, dl-met, l-thr, l-trp, l- Val, and l-ile. Results of Exp. 2 indicate that a 13.5% CP:Lys diet may be sufficient to maintain maximal performance and N retention. Assuming a linear response between the ingested and absorbed N and the dietary CP:SID Lys content in the N balance, we calculated that pigs fed the 13.5% CP diet may retain 77% of the ingested N and 91% of the absorbed N. This may be close to the maximal efficiency of N utilization by the pig because the dietary AA profile approaches the ideal AA profile. However, this efficiency value may be overestimated because the N balance technique overestimates true N retention because of incomplete quantification of N losses (Quiniou et al., 1995). The AA profile in the diets was designed to match or to slightly exceed all AA requirements. Reducing the dietary CP:Lys level to 13.5% did not decrease performance, and thus, the SID ideal AA profile used in the present study (51% Ile:Lys, 100% Leu:Lys, 32% His:Lys, 61% Phe:Lys, and 95% Phe + Tyr:Lys) is sufficient to maximize growth. In young pigs, the rate of

6 642 Gloaguen et al. Arg synthesis may be limited, and low intakes of Arg may result in reduced performance (Southern and Baker, 1983; Edmonds et al., 1987). The current SID Arg:Lys requirement estimate is 46% in 11- to 25-kg pigs (NRC, 2012). The use of the 14.0% CP:Lys diet, which contained 42% SID Arg:Lys, did not result in reduced performance of pigs. This means that the SID Arg:Lys requirement may not be greater than 42% in 10- to 20- kg pigs. In addition to dietary IAA, pigs need N for the synthesis of AA. In Exp. 2, the 13.0% CP:Lys diet may be deficient in N, whereas the 14.0% CP:Lys diet restored performance through the addition of l-glu. The rate of synthesis of DAA may be limited by the availability of the dietary or metabolic N, originating from the deamination of AA. Part of the IAA supply will be catabolized, and N released is converted to urea or can be used for the synthesis of AA. Nevertheless, results of the present experiments showed that N from IAA covers only part of the dietary N requirement for the synthesis of DAA. In addition to IAA, DAA or a source of N must be provided in the diet to maintain protein deposition and growth. When the dietary CP content is decreased, N may become deficient because of the reduction in DAA and, although more IAA will be supplied, close to the requirement, the lower supply of N resulting from the deamination of excess IAA. The N requirement is generally expressed as a ratio of N provided by IAA to DAA (IAA N ) or as a ratio of N provided by IAA to total N (Wang and Fuller, 1989; Heger et al., 1998; Lenis et al., 1999). In Exp. 2, the IAAN:DAAN supplies are 44:56 and 40:60 when dietary CP:Lys is increased from 13.0% to 14.0%, respectively. In growing pigs, the optimum dietary IAA N has been estimated to be 45:55 (Wang and Fuller, 1989), 48:52 (Heger et al., 1998), and 50:50 (Lenis et al., 1999). The results from our study indicate that the N requirement may be even lower than that of Wang and Fuller (1989). In addition, these authors considered Arg to be a DAA, which results in a lower IAA N value. In Exp. 2, the N requirement was estimated between 13.0% and 14.0% CP:Lys. This also may explain the reduction in ADG with the 14.0% and 12.7% CP diets in Exp. 1, which may have been deficient in N. These results also indicate that dietary N may be limiting for growth before Arg when dietary CP is reduced. After the most limiting AA in a cereal soybean meal diet (Lys, Thr, Met, and Trp), the sequence of the next-limiting AA for growth may be Val > [His, Ile, Leu, Phe (+Tyr)] > N > Arg. Expressing the N requirement as IAA N does not take into account that N can be provided by nonprotein N intake or that N can be available through deamination of IAA (Lenis et al., 1999). It is also assumed that the optimum IAA N is independent of the CP level. However, in Exp. 2, the 13.5% CP:Lys diet that provided 45:55 IAA N allowed pigs to maintain performance, whereas the 13.0% CP:Lys diet that provided a similar IAA N ratio resulted in a reduced feed efficiency. The 13.5% CP:Lys diet contains greater levels of Arg and DAA, which are a source of N for AA synthesis, than the 13.0% CP:Lys diet. Therefore, the use of IAA N to estimate the N requirement is inadequate. We propose that the dietary N requirement should take into account nonprotein N, DAA, and IAA and should be expressed as N:Lys, as the minimal dietary N:Lys level that maintains growth in pigs. Moreover, to satisfy N requirement, bioavailable N of the feed should be known. Dietary N is not 100% digestible, and the digestibility of N varies according to composition of the diet. The formulation of the feed on a digestible basis rather than total N may be more appropriate. For AA, the ileal digestibility (on an apparent or standardized basis) provides a more accurate estimate of AA bioavailability than apparent total tract digestibility because the absorption of AA in the lower gut is negligible and because of microbial AA synthesis. For N, Columbus et al. (2012) recently demonstrated that the infusion of casein and urea in the cecum of growing pigs fed a Val-limiting diet increased N balance and urea flux, indicating that the N absorbed from the lower gut in the form of ammonia can be used for the synthesis of DAA. Therefore, the apparent or standardized ileal digestibility may underestimate N bioavailability. The optimal digestible N relative to SID Lys was between 1.91% and 2.04% in Exp. 2. Chung and Baker (1992) showed that a Glu, Pro, and Gly mixture in a complete purified AA diet is an efficient source of N to maximize growth in 10-kg pigs. Consequently, we formulated the 13.0% and 14.0% CP:Lys diets with a minimum of Pro:Lys, Gly:Lys, and Glu:Lys to ensure maximal performance in Exp. 2. In growing pigs, Tyr, Cys, and Arg are commonly considered as conditionally IAA because their synthesis can be limited by dietary precursors, resulting in reduced growth (NRC, 2012). Recently, Powell et al. (2011) reported that a corn soybean meal diet with a CP:Lys content of 13.0% required the supplementation of Gly or Gly + Arg to maintain maximal growth in 20- to 40- kg pigs. This result was not a consequence of N deficiency because the supplementation of Glu did not improve performance. Therefore, Gly may be considered as a conditionally IAA in pigs. Wu (2010) indicated that DAA such as Gln, Glu, Pro, and Arg regulate metabolic pathways. Because dietary DAA are substantially catabolized by the small intestine, the ideal protein for growth should include all DAA (Wu, 2010). Finally, some of the traditionally classified DAA may be limiting for growth in very low-cp diets. In conclusion, the present study shows that it is possible to formulate very low-cp diets that maintain the

7 Low crude protein diets for piglets 643 growth of 10- to 20-kg pigs as long as AA needs are satisfied. In addition to the usual free AA (Lys, Met, Thr, and Trp), the use of free Val, Leu, Ile, His, and Phe enables a 4% unit reduction in dietary CP relative to SID Lys, and soybean meal can be totally replaced by cereals and free AA. A further reduction in dietary CP level reduces feed efficiency, indicating that the supply of N may be a limiting factor. The results indicate that a SID AA supply of 51% Ile:Lys, 100% Leu:Lys, 32% His:Lys, 61% Phe:Lys, 95% Phe + Tyr:Lys, and 42% Arg:Lys is sufficient to maintain growth in pigs. LITERATURE CITED AFNOR NF V Animal feeding stuffs. Determination of nitrogen content. Combustion method (DUMAS). Assoc. Fr. Normalisation, Paris. Apper-Bossard, E., J. L. Peyraud, and J. Y. Dourmad Effet du bilan électrolytique de la ration sur l équilibre acido-basique et les performances zootechniques des animaux domestiques à fort niveau de production. 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